Compounds, compositions and methods

ABSTRACT

Compounds useful for treating cellular proliferative diseases and disorders by modulating the activity of KSP are disclosed.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/582,424, filed Oct. 20, 2009, which is a continuation of U.S.application Ser. No. 12/142,621, filed Jun. 19, 2008, issued as U.S.Pat. No. 7,629,477, which is a continuation of U.S. application Ser. No.11/154,989, filed Jun. 15, 2005, issued as U.S. Pat. No. 7,491,746,which is a continuation of U.S. application Ser. No. 10/412,712, filedApr. 11, 2003, issued as U.S. Pat. No. 6,924,376, which claims priorityfrom U.S. Provisional Application Nos. 60/373,454, filed Apr. 17, 2002and 60/410,682, filed Sep. 13, 2002, each of which are incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to compounds which are inhibitors of the mitotickinesin KSP and are useful in the treatment of cellular proliferativediseases, for example cancer, hyperplasias, restenosis, cardiachypertrophy, immune disorders, fungal disorders, and inflammation.

BACKGROUND OF THE INVENTION

Among the therapeutic agents used to treat cancer are the taxanes andvinca alkaloids, which act on microtubules. Microtubules are the primarystructural element of the mitotic spindle. The mitotic spindle isresponsible for distribution of replicate copies of the genome to eachof the two daughter cells that result from cell division. It is presumedthat disruption of the mitotic spindle by these drugs results ininhibition of cancer cell division, and induction of cancer cell death.However, microtubules form other types of cellular structures, includingtracks for intracellular transport in nerve processes. Because theseagents do not specifically target mitotic spindles, they have sideeffects that limit their usefulness.

Improvements in the specificity of agents used to treat cancer is ofconsiderable interest because of the therapeutic benefits which would berealized if the side effects associated with the administration of theseagents could be reduced. Traditionally, dramatic improvements in thetreatment of cancer are associated with identification of therapeuticagents acting through novel mechanisms. Examples of this include notonly the taxanes, but also the camptothecin class of topoisomerase Iinhibitors. From both of these perspectives, mitotic kinesins areattractive targets for new anti-cancer agents.

Mitotic kinesins are enzymes essential for assembly and function of themitotic spindle, but are not generally part of other microtubulestructures, such as in nerve processes. Mitotic kinesins play essentialroles during all phases of mitosis. These enzymes are “molecular motors”that transform energy released by hydrolysis of ATP into mechanicalforce which drives the directional movement of cellular cargoes alongmicrotubules. The catalytic domain sufficient for this task is a compactstructure of approximately 340 amino acids. During mitosis, kinesinsorganize microtubules into the bipolar structure that is the mitoticspindle. Kinesins mediate movement of chromosomes along spindlemicrotubules, as well as structural changes in the mitotic spindleassociated with specific phases of mitosis. Experimental perturbation ofmitotic kinesin function causes malformation or dysfunction of themitotic spindle, frequently resulting in cell cycle arrest and celldeath.

Among the mitotic kinesins which have been identified is KSP. KSPbelongs to an evolutionarily conserved kinesin subfamily of plusend-directed microtubule motors that assemble into bipolar homotetramersconsisting of antiparallel homodimers. During mitosis KSP associateswith microtubules of the mitotic spindle. Microinjection of antibodiesdirected against KSP into human cells prevents spindle pole separationduring prometaphase, giving rise to monopolar spindles and causingmitotic arrest and induction of programmed cell death. KSP and relatedkinesins in other, non-human, organisms, bundle antiparallelmicrotubules and slide them relative to one another, thus forcing thetwo spindle poles apart. KSP may also mediate in anaphase B spindleelongation and focussing of microtubules at the spindle pole.

Human KSP (also termed HsEg5) has been described (Blangy, et al., Cell,83:1159-69 (1995); Whitehead, et al., Arthritis Rheum., 39:1635-42(1996); Galgio et al., J. Cell Biol., 135:339-414 (1996); Blangy, etal., J Biol. Chem., 272:19418-24 (1997); Blangy, et al., Cell MotilCytoskeleton, 40:174-82 (1998); Whitehead and Rattner, J. Cell Sci.,111:2551-61 (1998); Kaiser, et al., JBC 274:18925-31 (1999); GenBankaccession numbers: X85137, NM004523 and U37426), and a fragment of theKSP gene (TRIP5) has been described (Lee, et al., Mol Endocrinol.,9:243-54 (1995); GenBank accession number L40372). Xenopus KSP homologs(Eg5), as well as Drosophila KLP61 F/KRP1 30 have been reported.

Mitotic kinesins, including KSP, are attractive targets for thediscovery and development of novel antimitotic chemotherapeutics.Accordingly, it is an object of the present invention to providecompounds, compositions and methods useful in the inhibition of KSP.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides compounds that can be used to treat cellular proliferativediseases. The compounds are KSP inhibitors, particularly human KSPinhibitors. The present invention also provides compositions comprisingsuch compounds, and methods utilizing such compounds or compositions,which can be used to treat cellular proliferative diseases.

In one aspect, the invention relates to methods for treating cellularproliferative diseases, and for treating disorders by inhibiting theactivity of KSP. The methods employ compounds represented by Formula I:

wherein:

R₁ is chosen from hydrogen, optionally substituted alkyl-, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heteroaryl-, and optionally substituted heteroaralkyl-;

R₂ and R_(2′) are independently chosen from hydrogen, optionallysubstituted alkyl-, optionally substituted alkoxy, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heteroaryl-, and optionally substituted heteroaralkyl-; orR₂ and R_(2′) taken together form an optionally substituted 3- to7-membered ring;

R₁₂ is selected from the group consisting of optionally substitutedimidazolyl, optionally substituted imidazolinyl, —NHR₄; —N(R₄)(COR₃);—N(R₄)(SO₂R_(3a)); and —N(R₄)(CH₂R_(3b));

R₃ is chosen from hydrogen, optionally substituted alkyl-, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heteroaryl-, optionally substituted heteroaralkyl-, R₁₅O—and R₁₇—NH—;

R_(3a) is chosen from optionally substituted alkyl-, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heteroaryl-, optionally substituted heteroaralkyl-, andR₁₇—NH—;

R_(3b) is chosen from hydrogen, optionally substituted alkyl-,optionally substituted aryl-, optionally substituted aralkyl-,optionally substituted heteroaryl-, and optionally substitutedheteroaralkyl-;

R₄ is chosen from hydrogen, optionally substituted alkyl-, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heterocyclyl-, and optionally substituted heteroaralkyl-;

R₅, R₆, R₇ and R₈ are independently chosen from hydrogen, acyl,optionally substituted alkyl-, optionally substituted alkoxy, halogen,hydroxyl, nitro, cyano, dialkylamino, alkylsulfonyl-, alkylsulfonamido-,alkylthio-, carboxyalkyl-, carboxamido-, aminocarbonyl-, optionallysubstituted aryl and optionally substituted heteroaryl-;

R₁₅ is chosen from optionally substituted alkyl-, optionally substitutedaryl-, optionally substituted aralkyl-, optionally substitutedheteroaryl-, and optionally substituted heteroaralkyl-; and

R₁₇ is hydrogen, optionally substituted alkyl-, optionally substitutedaryl-, optionally substituted aralkyl-,

optionally substituted heteroaryl-, or optionally substitutedhetero-aralkyl-, including single stereoisomers, mixtures ofstereoisomers;

a pharmaceutically acceptable salt of a compound of Formula I;

a pharmaceutically acceptable solvate of a pharmaceutically acceptablesolvate of a compound of Formula I; or

a pharmaceutically acceptable solvate of a pharmaceutically acceptablesalt of a compound of Formula I.

According to one embodiment, when either R₂ or R_(2′) is hydrogen, theother is not hydrogen. In another embodiment, R₂ and R_(2′) are eachhydrogen; and R₁ is chosen from optionally substituted aryl, optionallysubstituted aralkyl, optionally substituted heteroaryl, and optionallysubstituted heteroaralkyl, provided however, that R₁ is not substitutedphenyl.

In one aspect, the invention relates to methods for treating cellularproliferative diseases and other disorders that can be treated byinhibiting KSP by the administration of a therapeutically effectiveamount of a compound of Formula I; a pharmaceutically acceptable salt ofa compound of Formula I; or a pharmaceutically acceptable solvate of apharmaceutically acceptable salt of a compound of Formula I. thereof.Such diseases and disorders include cancer, hyperplasia, restenosis,cardiac hypertrophy, immune disorders, fungal disorders andinflammation.

In another aspect, the invention relates to compounds useful ininhibiting KSP kinesin. The compounds have the structures shown above inFormula I; a pharmaceutically acceptable salt of a compound of FormulaI; or a pharmaceutically acceptable solvate of a pharmaceuticallyacceptable salt of a compound of Formula I. The invention also relatesto pharmaceutical compositions containing a therapeutically effectiveamount of a compound of Formula I; a pharmaceutically acceptable salt ofa compound of Formula I; or a pharmaceutically acceptable solvate of apharmaceutically acceptable salt of a compound of Formula I, admixedwith at least one pharmaceutical excipient. In another aspect, thecomposition further comprises a chemotherapeutic agent other than acompound of the present invention.

In an additional aspect, the present invention provides methods ofscreening for compounds that will bind to a KSP kinesin, for examplecompounds that will displace or compete with the binding of a compoundof the invention. The methods comprise combining a labeled compound ofthe invention, a KSP kinesin, and at least one candidate agent anddetermining the binding of the candidate agent to the KSP kinesin.

In a further aspect, the invention provides methods of screening formodulators of KSP kinesin activity. The methods comprise combining acompound of the invention, a KSP kinesin, and at least one candidateagent and determining the effect of the candidate agent on the KSPkinesin activity.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be better understood by reference to the followingdescription taken in conjunction with the accompanying drawings.

FIGS. 1 and 2 represent a XRPD and MDSC scan, respectively, of a saltprepared according to Preparation A.

FIG. 3 represents an XRPD scan of a salt prepared according toPreparation B.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise. The following abbreviations and terms have the indicatedmeanings throughout:

Ac=acetyl

Boc=t-butyloxy carbonyl

Bu=butyl

c-=cyclo

CBZ=carbobenzoxy=benzyloxycarbonyl

DCM=dichloromethane=methylene chloride=CH₂Cl₂

DIEA=N,N-diisopropylethylamine

DMF=N,N-dimethylformamide

DMSO=dimethyl sulfoxide

Et=ethyl

HBTU=O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate

HMDS=hexamethyldisilazane

HOAc=acetic acid

IPA=isopropyl alcohol

Me=methyl

Ph=phenyl

Py=pyridine

rt=room temperature

sat'd=saturated

s-=secondary

t-=tertiary

TEA=triethylamine

TFA=trifluoroacetic acid

THF=tetrahydrofuran

Tf=triflate

Alkyl is intended to include linear, branched, or cyclic aliphatichydrocarbon structures and combinations thereof, which structures may besaturated or unsaturated. Lower-alkyl refers to alkyl groups of from 1to 5 carbon atoms, preferably from 1 to 4 carbon atoms. Examples oflower-alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s-and t-butyl and the like. Preferred alkyl groups are those of C₂₀ orbelow. More preferred alkyl groups are those of C₁₃ or below. Cycloalkylis a subset of alkyl and includes cyclic aliphatic hydrocarbon groups offrom 3 to 13 carbon atoms. Examples of cycloalkyl groups includec-propyl, c-butyl, c-pentyl, norbornyl, adamantyl and the like.Cycloalkyl-alkyl- is another subset of alkyl and refers to cycloalkylattached to the parent structure through a non-cyclic alkyl. Examples ofcycloalkyl-alkyl- include cyclohexylmethyl, cyclopropylmethyl,cyclohexylpropyl, and the like. In this application, alkyl includesalkanyl, alkenyl and alkynyl residues; it is intended to include vinyl,allyl, isoprenyl and the like. Alkylene-, alkenylene-, and alkynylene-are other subsets of alkyl, including the same residues as alkyl, buthaving two points of attachment within a chemical structure. Examples ofalkylene include ethylene (—CH₂CH₂—), propylene (—CH₂CH₂CH₂—),dimethylpropylene (—CH₂C(CH₃)₂CH₂—) and cyclohexylpropylene(—CH₂CH₂CH(C₆H₁₃)—). Likewise, examples of alkenylene include ethenylene(—CH═CH—), propenylene (—CH═CH—CH₂—), and cyclohexylpropenylene(—CH═CHCH(C₆H₁₃)—). Examples of alkynylene include ethynylene (—C═C—)and propynylene (—CH═CH—CH₂—). When an alkyl residue having a specificnumber of carbons is named, all geometric isomers having that number ofcarbons are intended to be encompassed; thus, for example, “butyl” ismeant to include n-butyl, sec-butyl, isobutyl and t-butyl; “propyl”includes n-propyl, isopropyl, and c-propyl.

Alkoxy or alkoxyl refers to an alkyl group, preferably including from 1to 8 carbon atoms, of a straight, branched, or cyclic configuration, ora combination thereof, attached to the parent structure through anoxygen (i.e., the group alkyl-O—). Examples include methoxy-, ethoxy-,propoxy-, isopropoxy-, cyclopropyloxy-, cyclohexyloxy- and the like.Lower-alkoxy refers to alkoxy groups containing one to four carbons.

Acyl refers to groups of from 1 to 8 carbon atoms of a straight,branched, or cyclic configuration or a combination thereof, attached tothe parent structure through a carbonyl functionality. Such groups maybe saturated or unsaturated, and aliphatic or aromatic. One or morecarbons in the acyl residue may be replaced by nitrogen, oxygen orsulfur as long as the point of attachment to the parent remains at thecarbonyl. Examples include acetyl, benzoyl, propionyl, isobutyryl,t-butoxycarbonyl, benzyloxycarbonyl and the like. Lower-acyl refers toacyl groups containing one to four carbons.

Amino refers to the group —NH₂. The term “substituted amino” refers tothe group —NHR or —NRR where each R is independently selected from thegroup: optionally substituted alkyl, optionally substituted alkoxy,optionally substituted amino carbonyl, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted heterocyclyl,acyl, alkoxycarbonyl, sulfanyl, sulfinyl and sulfonyl, e.g.,diethylamino, methylsulfonylamino, furanyl-oxy-sulfonamino.

Aminocarbonyl—refers to the group —NR^(c)COR^(b), —NR^(c)CO₂R^(b), or—NR^(c)CONR^(b)R^(c), where

R^(b) is H or optionally substituted C₁-C₆ alkyl, aryl, heteroaryl,aryl-C₁-C₄ alkyl-, or heteroaryl-C₁-C₄ alkyl- group; and

R^(c) is hydrogen or C₁-C₄ alkyl; and

where each optionally substituted R^(b) group is independentlyunsubstituted or substituted with one or more substituents independentlyselected from C₁-C₄ alkyl, aryl, heteroaryl, aryl-C₁-C₄ alkyl-,heteroaryl-C₁-C₄ alkyl-, C₁-C₄ haloalkyl, —OC₁-C₄ alkyl, —OC₁-C₄alkylphenyl, —C₁-C₄ alkyl-OH, —OC₁-C₄ haloalkyl, halogen, —OH, —NH₂,—C₁-C₄ alkyl-NH₂, —N(C₁-C₄ alkyl)(C₁-C₄ alkyl), —NH(C₁-C₄ alkyl),—N(C₁-C₄ alkyl)(C₁-C₄ alkylphenyl), —NH(C₁-C₄ alkylphenyl), cyano,nitro, oxo (as a substitutent for heteroaryl), —CO₂H, —C(O)OC₁-C₄ alkyl,—CON(C₁-C₄ alkyl)(C₁-C₄ alkyl), —CONH(C₁-C₄ alkyl), —CONH₂,—NHC(O)(C₁-C₄ alkyl), —NHC(O)(phenyl), —N(C₁-C₄ alkyl)C(O)(C₁-C₄ alkyl),—N(C₁-C₄ alkyl)C(O)(phenyl), —C(O)C₁-C₄ alkyl, —C(O)C₁-C₄ phenyl,—C(O)C₁-C₄ haloalkyl, —OC(O)C₁-C₄ alkyl, —SO₂(C₁-C₄ alkyl),—SO₂(phenyl), —SO₂(C₁-C₄ haloalkyl), —SO₂NH₂, —SO₂NH(C₁-C₄ alkyl),—SO₂NH(phenyl), —NHSO₂(C₁-C₄ alkyl), —NHSO₂(phenyl), and —NHSO₂(C₁-C₄haloalkyl).

Antimitotic refers to a drug for inhibiting or preventing mitosis, forexample, by causing metaphase arrest. Some antitumour drugs blockproliferation and are considered antimitotics.

Aryl and heteroaryl mean a 5- or 6-membered aromatic or heteroaromaticring containing 0 or 1-4 heteroatoms, respectively, selected from O, N,or S; a bicyclic 9- or 10-membered aromatic or heteroaromatic ringsystem containing 0 or 1-4 (or more) heteroatoms, respectively, selectedfrom O, N, or S; or a tricyclic 12- to 14-membered aromatic orheteroaromatic ring system containing 0 or 1-4 (or more) heteroatoms,respectively, selected from O, N, or S. The aromatic 6- to 14-memberedcarbocyclic rings include, e.g., phenyl, naphthyl, indanyl, tetralinyl,and fluorenyl and the 5- to 10-membered aromatic heterocyclic ringsinclude, e.g., imidazolyl, pyridinyl, indolyl, thienyl, benzopyranonyl,thiazolyl, furanyl, benzimidazolyl, quinolinyl, isoquinolinyl,quinoxalinyl, pyrimidinyl, pyrazinyl, tetrazolyl and pyrazolyl.

Aralkyl—refers to a residue in which an aryl moiety is attached to theparent structure via an alkyl residue. Examples include benzyl,phenethyl, phenylvinyl, phenylallyl and the like. Heteroaralkyl—refersto a residue in which a heteroaryl moiety is attached to the parentstructure via an alkyl residue. Examples include furanylmethyl,pyridinylmethyl, pyrimidinylethyl and the like.

Aralkoxy—refers to the group —O-aralkyl. Similarly,heteroaralkoxy—refers to the group —O-heteroaralkyl; aryloxy- refers tothe group —O-aryl; acyloxy- refers to the group —O-acyl; heteroaryloxy-refers to the group —O-heteroaryl; and heterocyclyloxy- refers to thegroup —O-heterocyclyl (i.e., aralkyl, heteroaralkyl, aryl, acyl,heterocyclyl, or heteroaryl is attached to the parent structure throughan oxygen).

Carboxyalkyl—refers to the group -alkyl-COOH.

Carboxamido refers to the group —CONR^(b)R^(c), where

R^(b) is H or optionally substituted C₁-C₆ alkyl, aryl, heteroaryl,aryl-C₁-C₄ alkyl-, or heteroaryl-C₁-C₄ alkyl- group; and

R^(c) is hydrogen or C₁-C₄ alkyl; and

where each optionally substituted R^(b) group is independentlyunsubstituted or substituted with one or more substituents independentlyselected from C₁-C₄ alkyl, aryl, heteroaryl, aryl-C₁-C₄ alkyl-,heteroaryl-C₁-C₄ alkyl-, C₁-C₄ haloalkyl, —OC₁-C₄ alkyl, —OC₁-C₄alkylphenyl, —C₁-C₄ alkyl-OH, —OC₁-C₄ haloalkyl, halogen, —OH, —NH₂,—C₁-C₄ alkyl-NH₂, —N(C₁-C₄ alkyl)(C₁-C₄ alkyl), —NH(C₁-C₄ alkyl),—N(C₁-C₄ alkyl)(C₁-C₄ alkylphenyl), —NH(C₁-C₄ alkylphenyl), cyano,nitro, oxo (as a substitutent for heteroaryl), —CO₂H, —C(O)OC₁-C₄ alkyl,—CON(C₁-C₄ alkyl)(C₁-C₄ alkyl), —CONH(C₁-C₄ alkyl), —CONH₂,—NHC(O)(C₁-C₄ alkyl), —NHC(O)(phenyl), —N(C₁-C₄ alkyl)C(O)(C₁-C₄ alkyl),—N(C₁-C₄ alkyl)C(O)(phenyl), —C(O)C₁-C₄ alkyl, —C(O)C₁-C₄ phenyl,—C(O)C₁-C₄ haloalkyl, —OC(O)C₁-C₄ alkyl, —SO₂(C₁-C₄ alkyl),—SO₂(phenyl), —SO₂(C₁-C₄ haloalkyl), —SO₂NH₂, —SO₂NH(C₁-C₄ alkyl),—SO₂NH(phenyl), —NHSO₂(C₁-C₄ alkyl), —NHSO₂(phenyl), and —NHSO₂(C₁-C₄haloalkyl).

Halogen or halo refers to fluorine, chlorine, bromine or iodine.Fluorine, chlorine and bromine are preferred. Dihaloaryl, dihaloalkyl,trihaloaryl etc. refer to aryl and alkyl substituted with the designatedplurality of halogens (here, 2, 2 and 3, respectively), but notnecessarily a plurality of the same halogen; thus4-chloro-3-fluorophenyl is within the scope of dihaloaryl.

Heterocyclyl means a cycloalkyl or aryl residue in which one to four ofthe carbons is replaced by a heteroatom such as oxygen, nitrogen orsulfur. Examples of heterocycles that fall within the scope of theinvention include azetidinyl, imidazolinyl, pyrrolidinyl, pyrazolyl,pyrrolyl, indolyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl,benzofuranyl, benzodioxanyl, benzodioxyl (commonly referred to asmethylenedioxyphenyl, when occurring as a substituent), tetrazolyl,morpholinyl, thiazolyl, pyridinyl, pyridazinyl, piperidinyl,pyrimidinyl, thienyl, furanyl, oxazolyl, oxazolinyl, isoxazolyl,dioxanyl, tetrahydrofuranyl and the like. “N-heterocyclyl” refers to anitrogen-containing heterocycle. The term heterocyclyl encompassesheteroaryl, which is a subset of heterocyclyl. Examples ofN-heterocyclyl residues include azetidinyl, 4-morpholinyl,4-thiomorpholinyl, 1-piperidinyl, 1-pyrrolidinyl, 3-thiazolidinyl,piperazinyl and 4-(3,4-dihydrobenzoxazinyl). Examples of substitutedheterocyclyl include 4-methyl-1-piperazinyl and 4-benzyl-1-piperidinyl.

A leaving group or atom is any group or atom that will, under thereaction conditions, cleave from the starting material, thus promotingreaction at a specified site. Suitable examples of such groups unlessotherwise specified are halogen atoms, mesyloxy,p-nitrobenzensulphonyloxy and tosyloxy groups.

Optional or optionally means that the subsequently described event orcircumstance may or may not occur, and that the description includesinstances where said event or circumstances occurs and instances inwhich it does not. For example, “optionally substituted alkyl” includes“alkyl” and “substituted alkyl” as defined herein. It will be understoodby those skilled in the art with respect to any group containing one ormore substituents that such groups are not intended to introduce anysubstitution or substitution patterns that are sterically impracticaland/or synthetically non-feasible and/or inherently unstable.

Substituted alkoxy refers to alkoxy wherein the alkyl constituent issubstituted (i.e., —O-(substituted alkyl)). One preferred substitutedalkoxy group is “polyalkoxy” or —O-(optionally substitutedalkylene)-(optionally substituted alkoxy), and includes groups such as—OCH₂CH₂OCH₃, and residues of glycol ethers such as polyethyleneglycol,and —O(CH₂CH₂O)_(x)CH₃, where x is an integer of about 2-20, preferablyabout 2-10, and more preferably about 2-5. Another preferred substitutedalkoxy group is hydroxyalkoxy or —OCH₂(CH₂)_(y)OH, where y is an integerof about 1-10, preferably about 1-4.

Substituted—alkyl, aryl, and heteroaryl, which includes the substitutedalkyl, aryl and heteroaryl moieties of any group containing anoptionally substituted alkyl, aryl and heteroaryl moiety (e.g., alkoxy,aralkyl and heteroaralkyl), refer respectively to alkyl, aryl, andheteroaryl wherein one or more (up to about 5, preferably up to about 3)hydrogen atoms are replaced by a substituent independently selected fromthe group:

—R^(a), —OR^(b), —O(C₁-C₂ alkyl)O— (as an aryl substituent), —SR^(b),—NR^(b)R^(c), halogen, cyano, nitro, —COR^(b), —CO₂R^(b),—CONR^(b)R^(c), —OCOR^(b), —OCO₂R^(b), —OCONR^(b)R^(c), —NR^(c)COR^(b),—NR^(c)CO₂R^(b), —NR^(c)CONR^(b)R^(c), —CO₂R^(b), —CONR^(b)R^(c),—NR^(c)COR^(b), —SOR^(a), —SO₂R^(a), —SO₂NR^(b)R^(c), and—NR^(c)SO₂R^(a),

where R^(a) is an optionally substituted C₁-C₆ alkyl, aryl, heteroaryl,aryl-C₁-C₄ alkyl-, or heteroaryl-C₁-C₄ alkyl- group,

R^(b) is H or optionally substituted C₁-C₆ alkyl, aryl, heteroaryl,aryl-C₁-C₄ alkyl-, or heteroaryl-C₁-C₄ alkyl- group;

R^(c) is hydrogen or C₁-C₄ alkyl;

where each optionally substituted R^(a) group and R^(b) group isindependently unsubstituted or substituted with one or more substituentsindependently selected from C₁-C₄ alkyl, aryl, heteroaryl, aryl-C₁-C₄alkyl-, heteroaryl-C₁-C₄ alkyl-, C₁-C₄ haloalkyl, —OC₁-C₄ alkyl, —OC₁-C₄alkylphenyl, —C₁-C₄ alkyl-OH, —OC₁-C₄ haloalkyl, halogen, —OH, —NH₂,—C₁-C₄ alkyl-NH₂, —N(C₁-C₄ alkyl)(C₁-C₄ alkyl), —NH(C₁-C₄ alkyl),—N(C₁-C₄ alkyl)(C₁-C₄ alkylphenyl), —NH(C₁-C₄ alkylphenyl), cyano,nitro, oxo (as a substitutent for heteroaryl), —CO₂H, —C(O)OC₁-C₄ alkyl,—CON(C₁-C₄ alkyl)(C₁-C₄ alkyl), —CONH(C₁-C₄ alkyl), —CONH₂,—NHC(O)(C₁-C₄ alkyl), —NHC(O)(phenyl), —N(C₁-C₄ alkyl)C(O)(C₁-C₄ alkyl),—N(C₁-C₄ alkyl)C(O)(phenyl), —C(O)C₁-C₄ alkyl, —C(O)C₁-C₄ phenyl,—C(O)C₁-C₄ haloalkyl, —OC(O)C₁-C₄ alkyl, —SO₂(C₁-C₄ alkyl),—SO₂(phenyl), —SO₂(C₁-C₄ haloalkyl), —SO₂NH₂, —SO₂NH(C₁-C₄ alkyl),—SO₂NH(phenyl), —NHSO₂(C₁-C₄ alkyl), —NHSO₂(phenyl), and —NHSO₂(C₁-C₄haloalkyl).

Sulfanyl refers to the groups: —S-(optionally substituted alkyl),—S-(optionally substituted aryl), —S-(optionally substitutedheteroaryl), and —S-(optionally substituted heterocyclyl).

Sulfanyl refers to the groups: —S(O)—H, —S(O)-(optionally substitutedalkyl), —S(O)-optionally substituted aryl), —S(O)-(optionallysubstituted heteroaryl), —S(O)-(optionally substituted heterocyclyl);and —S(O)-(optionally substituted amino).

Sulfonyl refers to the groups: —S(O₂)—H, —S(O₂)-(optionally substitutedalkyl), —S(O₂)-optionally substituted aryl), —S(O₂)-(optionallysubstituted heteroaryl), —S(O₂)-(optionally substituted heterocyclyl),—S(O₂)-(optionally substituted alkoxy), —S(O₂)-optionally substitutedaryloxy), —S(O₂)-(optionally substituted heteroaryloxy),—S(O₂)-(optionally substituted heterocyclyloxy); and —S(O₂)-(optionallysubstituted amino).

Pharmaceutically acceptable salts refers to those salts that retain thebiological effectiveness of the free compound and that are notbiologically or otherwise undesirable, formed with a suitable acid orbase, and includes pharmaceutically acceptable acid addition salts andbase addition salts. Pharmaceutically acceptable acid addition saltsinclude those derived from inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and those derived from organic acids such as acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike.

Pharmaceutically acceptable base addition salts include those derivedfrom inorganic bases such as sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, aluminum salts andthe like. Particularly preferred are the ammonium, potassium, sodium,calcium, and magnesium salts. Base addition salts also include thosederived from pharmaceutically acceptable organic non-toxic bases,including salts of primary, secondary, and tertiary amines, substitutedamines including naturally occurring substituted amines, cyclic aminesand basic ion exchange resins, such as isopropylamine, trimethylamine,diethylamine, triethylamine, tripropylamine, and ethanolamine.

Protecting group has the meaning conventionally associated with it inorganic synthesis, i.e. a group that selectively blocks one or morereactive sites in a multifunctional compound such that a chemicalreaction can be carried out selectively on another unprotected reactivesite and such that the group can readily be removed after the selectivereaction is complete. A variety of protecting groups are disclosed, forexample, in T. H. Greene and P. G. M. Wuts, Protective Groups in OrganicSynthesis, Third Edition, John Wiley & Sons, New York (1999), which isincorporated herein by reference in its entirety. For example, a hydroxyprotected form is where at least one of the hydroxyl groups present in acompound is protected with a hydroxy protecting group. Likewise, aminesand other reactive groups may similarly be protected.

Solvate refers to the compound formed by the interaction of a solventand a compound of Formula I or salt thereof. Suitable solvates of thecompounds of the Formula I are pharmaceutically acceptable solvates,such as hydrates, including monohydrates and hemi-hydrates.

Many of the compounds described herein contain one or more asymmetriccenters (e.g. the carbon to which R₂ and R_(2′) are attached where R₂differs from R_(2′)) and may thus give rise to enantiomers,diastereomers, and other stereoisomeric forms that may be defined, interms of absolute stereochemistry, as (R)- or (S)-. The presentinvention is meant to include all such possible isomers, includingracemic mixtures, optically pure forms and intermediate mixtures.Optically active (R)- and (S)-isomers may be prepared using chiralsynthons or chiral reagents, or resolved using conventional techniques.When the compounds described herein contain olefinic double bonds orother centers of geometric asymmetry, and unless specified otherwise, itis intended that the compounds include both E and Z geometric isomers.Likewise, all tautomeric forms and rotational isomers are also intendedto be included.

When desired, the R- and S-isomers may be resolved by methods known tothose skilled in the art, for example by formation of diastereoisomericsalts or complexes which may be separated, for example, bycrystallization; via formation of diastereoisomeric derivatives whichmay be separated, for example, by crystallization, gas-liquid or liquidchromatography; selective reaction of one enantiomer with anenantiomer-specific reagent, for example enzymatic oxidation orreduction, followed by separation of the modified and unmodifiedenantiomers; or gas-liquid or liquid chromatography in a chiralenvironment, for example on a chiral support, such as silica with abound chiral ligand or in the presence of a chiral solvent. It will beappreciated that where the desired enantiomer is converted into anotherchemical entity by one of the separation procedures described above, afurther step may be required to liberate the desired enantiomeric form.Alternatively, specific enantiomer may be synthesized by asymmetricsynthesis using optically active reagents, substrates, catalysts orsolvents, or by converting one enantiomer to the other by asymmetrictransformation.

Compounds of the Present Invention

The present invention is directed to a class of novel compounds, whichcan be described as benzopyran-4-ones or chromen-4-ones, that areinhibitors of one or more mitotic kinesins. By inhibiting mitotickinesins, but not other kinesins (e.g., transport kinesins), specificinhibition of cellular proliferation is accomplished. While notintending to be bound by any theory, the present invention capitalizeson the finding that perturbation of mitotic kinesin function causesmalformation or dysfunction of mitotic spindles, frequently resulting incell cycle arrest and cell death. According to one embodiment of theinvention, the compounds described herein inhibit the mitotic kinesin,KSP. In another embodiment, the compounds inhibit the mitotic kinesin,KSP, as well as modulating one or more of the human mitotic kinesinsselected from the group consisting of HSET (see, U.S. Pat. No.6,361,993, which is incorporated herein by reference); MCAK (see, U.S.Pat. No. 6,331,424, which is incorporated herein by reference); CENP-E(see, PCT Publication No. WO 99/13061, which is incorporated herein byreference); Kif4 (see, U.S. Pat. No. 6,440,684, which is incorporatedherein by reference); MKLP1 (see, U.S. Pat. No. 6,448,025, which isincorporated herein by reference); Kif15 (see, U.S. Pat. No. 6,355,466,which is incorporated herein by reference); Kid (see, U.S. Pat. No.6,387,644, which is incorporated herein by reference); Mppl, CMKrp,KinI-3 (see, U.S. Pat. No. 6,461,855, which is incorporated herein byreference); Kip3a (see, PCT Publication No. WO 01/96593, which isincorporated herein by reference); Kip3d (see, U.S. Pat. No. 6,492,151,which is incorporated herein by reference); and RabK6.

The methods of inhibiting a human KSP kinesin comprise contacting aninhibitor of the invention with a kinesin, particularly a human kinesin,preferably human KSP or fragments and variants thereof. The inhibitioncan be of the ATP hydrolysis activity of the KSP kinesin and/or themitotic spindle formation activity, such that the mitotic spindles aredisrupted. Meiotic spindles may also be disrupted.

An object of the present invention is to develop inhibitors of mitotickinesins, in particular KSP and especially human KSP, for the treatmentof disorders associated with cell proliferation. Traditionally, dramaticimprovements in the treatment of cancer, one type of cellularproliferative disorder, have been associated with identification oftherapeutic agents acting through novel mechanisms. Examples of thisinclude not only the taxane class of agents that appear to act onmicrotubule formation, but also the camptothecin class of topoisomeraseI inhibitors. The compounds, compositions and methods described hereincan differ in their selectivity and are preferably used to treatdiseases of cellular proliferation, including, but not limited tocancer, hyperplasias, restenosis, cardiac hypertrophy, immune disorders,fungal disorders and inflammation.

Accordingly, the present invention relates to methods employingcompounds represented by Formula I:

wherein:

R₁ is chosen from hydrogen, optionally substituted alkyl-, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heteroaryl-, and optionally substituted heteroaralkyl-;

R₂ and R_(2′) are independently chosen from hydrogen, optionallysubstituted alkyl-, optionally substituted alkoxy, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heteroaryl-, and optionally substituted heteroaralkyl-; orR₂ and R_(2′) taken together form an optionally substituted 3- to7-membered ring;

R₁₂ is selected from the group consisting of optionally substitutedimidazolyl, optionally substituted imidazolinyl, —NHR₄; —N(R₄)(COR₃);—N(R₄)(SO₂R_(3a)); and —N(R₄)(CH₂R_(3b));

R₃ is chosen from hydrogen, optionally substituted alkyl-, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heteroaryl-, optionally substituted heteroaralkyl-, R₁₅O—and R₁₇—NH—;

R_(3a) is chosen from optionally substituted alkyl-, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heteroaryl-, optionally substituted heteroaralkyl-, andR₁₇—NH—;

R_(3b) is chosen from hydrogen, optionally substituted alkyl-,optionally substituted aryl-, optionally substituted aralkyl-,optionally substituted heteroaryl-, and optionally substitutedheteroaralkyl-;

R₄ is chosen from hydrogen, optionally substituted alkyl-, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heterocyclyl-, and optionally substituted heteroaralkyl-;

R₅, R₆, R₇ and R₈ are independently chosen from hydrogen, acyl,optionally substituted alkyl-, optionally substituted alkoxy, halogen,hydroxyl, nitro, cyano, dialkylamino, alkylsulfonyl-, alkylsulfonamido-,alkylthio-, carboxyalkyl-, carboxamido-, aminocarbonyl-, optionallysubstituted aryl and optionally substituted heteroaryl-;

R₁₅ is chosen from optionally substituted alkyl-, optionally substitutedaryl-, optionally substituted aralkyl-, optionally substitutedheteroaryl-, and optionally substituted heteroaralkyl-, and

R₁₇ is hydrogen, optionally substituted alkyl-, optionally substitutedaryl-, optionally substituted aralkyl-, optionally substitutedheteroaryl-, or optionally substituted heteroaralkyl-, including singlestereoisomers, mixtures of stereoisomers;

a pharmaceutically acceptable salt of a compound of Formula I;

a pharmaceutically acceptable solvate of a pharmaceutically acceptablesolvate of a compound of Formula I;

or a pharmaceutically acceptable solvate of a pharmaceuticallyacceptable salt of a compound of Formula I.

When R₁₂ is an imidazole, R₁₂ has the formula:

wherein

R₉ is chosen from hydrogen, optionally substituted C₁-C₈ alkyl,optionally substituted aryl, optionally substituted aryl-C₁-C₄-alkyl-,optionally substituted heteroaryl-C₁-C₄-alkyl-, optionally substitutedaryl-C₁-C₄-alkoxy-, optionally substituted heteroaryl-C₁-C₄-alkoxy-,optionally substituted heteroaryl-; and R₁₃ and R_(13′) areindependently hydrogen, optionally substituted C₁-C₈ alkyl, optionallysubstituted aryl, or optionally substituted aryl-C₁-C₄-alkyl-.

When R₁₂ is an imidazoline, R₁₂ has the formula

wherein

R₉ is chosen from hydrogen, optionally substituted C₁-C₈ alkyl,optionally substituted aryl, optionally substituted aryl-C₁-C₄-alkyl-,and optionally substituted heteroaryl-; and R₁₀, R_(10′), R₁₄, andR_(14′) are independently chosen from hydrogen, optionally substitutedC₁-C₈ alkyl, optionally substituted aryl, and optionally substitutedaryl-C₁-C₄-alkyl-.

In one embodiment, R₁ is chosen from hydrogen, optionally substitutedalkyl, optionally substituted aryl, optionally substituted aralkyl,optionally substituted heteroaryl, and optionally substitutedheteroaralkyl-;

R₂ and R_(2′) are independently chosen from hydrogen, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedaryl, optionally substituted aralkyl, optionally substituted heteroaryl,and optionally substituted heteroaralkyl-; or R₂ and R_(2′) takentogether form an optionally substituted 3- to 7-membered ring, providedthat if either R₂ or R_(2′) is hydrogen, then the other is not hydrogen;

R₁₂ is selected from the group consisting of optionally substitutedimidazolyl, optionally substituted imidazolinyl, —NHR₄; —N(R₄)(COR₃);N(R₄)(SO₂R_(3a)); and —N(R₄)(CH₂R_(3b));

R₃ is chosen from hydrogen, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl, optionally substitutedheteroaryl, optionally substituted heteroaralkyl, R₁₅O— and R₁₇—NH—;

R_(3a) is chosen from optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl, optionally substitutedheteroaryl, optionally substituted heteroaralkyl, and R₁₇—NH—;

R_(3b) is chosen from hydrogen, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl, optionally substitutedheteroaryl, and optionally substituted heteroaralkyl-;

R₄ is chosen from hydrogen, optionally substituted alkyl-, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heterocyclyl-, and optionally substituted heteroaralkyl-;

R₅, R₆, R₇ and R₈ are independently chosen from hydrogen, acyl,optionally substituted alkyl, optionally substituted alkoxy, halogen,hydroxyl, nitro, cyano, dialkylamino, alkylsulfonyl, alkylsulfonamido,alkylthio, carboxyalkyl, carboxamido, aminocarbonyl, optionallysubstituted aryl and optionally substituted heteroaryl-;

R₁₅ is chosen from optionally substituted alkyl, optionally substitutedaryl, optionally substituted aralkyl, optionally substituted heteroaryl,and optionally substituted heteroaralkyl-; and

R₁₇ is chosen from hydrogen, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl, optionally substitutedheteroaryl, and optionally substituted heteroaralkyl, including singlestereoisomers, mixtures of stereoisomers;

a pharmaceutically acceptable salt of a compound of Formula I;

a pharmaceutically acceptable solvate of a pharmaceutically acceptablesolvate of a compound of Formula I; or

a pharmaceutically acceptable solvate of a pharmaceutically acceptablesalt of a compound of Formula I.

In another embodiment, R₂ and R_(2′) are hydrogen; and

R₁ is chosen from optionally substituted aryl, optionally substitutedaralkyl, optionally substituted heteroaryl, and optionally substitutedheteroaralkyl, provided however, that R₁ is not substituted phenyl;

R₁₂ is selected from the group consisting of optionally substitutedimidazolyl, optionally substituted imidazolinyl, —NHR₄; —N(R₄)(COR₃);—N(R₄)(SO₂R_(3a)); and —N(R₄)(CH₂R_(3b));

R₃ is chosen from hydrogen, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl, optionally substitutedheteroaryl, optionally substituted heteroaralkyl, R₁₅O— and R₁₇—NH—;

R_(3a) is chosen from optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl, optionally substitutedheteroaryl, optionally substituted heteroaralkyl, and R₁₇—NH—;

R_(3b) is chosen from hydrogen, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl, optionally substitutedheteroaryl, and optionally substituted heteroaralkyl-;

R₄ is chosen from hydrogen, optionally substituted alkyl-, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heterocyclyl-, and optionally substituted heteroaralkyl-;

R₅, R₆, R₇ and R₈ are independently chosen from hydrogen, acyl,optionally substituted alkyl, optionally substituted alkoxy, halogen,hydroxyl, nitro, cyano, dialkylamino, alkylsulfonyl, alkylsulfonamido,alkylthio, carboxyalkyl, carboxamido, aminocarbonyl, optionallysubstituted aryl and optionally substituted heteroaryl-;

R₁₅ is chosen from optionally substituted alkyl, optionally substitutedaryl, optionally substituted aralkyl, optionally substituted heteroaryl,and optionally substituted heteroaralkyl-; and

R₁₇ is chosen from hydrogen, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl, optionally substitutedheteroaryl, and optionally substituted heteroaralkyl, including singlestereoisomers, mixtures of stereoisomers;

a pharmaceutically acceptable salt of a compound of Formula I;

a pharmaceutically acceptable solvate of a pharmaceutically acceptablesolvate of a compound of Formula I; or

a pharmaceutically acceptable solvate of a pharmaceutically acceptablesalt of a compound of Formula I.

In a particularly preferred embodiment, where R₂ differs from R_(2′),the stereogenic center to which R₂ and R_(2′) are attached is of the Rconfiguration.

Nomenclature

The compounds of Formula I can be named and numbered in the manner(e.g., using AutoNom version 2.1 or ISIS-DRAW, each of which utilizesthe IUPAC system of nomenclature) described below. For example, thecompound:

i.e., the compound according to Formula I where R₁ is benzyl, R₂ ispropyl (particularly i-propyl), R_(2′) is hydrogen; R₁₂ is —N(R₄)(COR₃);R₃ is 3,4-dimethylphenyl-; R₄ is 3-aminopropyl-; R₅, R₆, and R₈ arehydrogen; and R₇ is cyano is namedN-(3-amino-propyl)-N-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-3,4-dimethyl-benzamide.

Likewise, the compound:

i.e., the compound according to Formula I where R₁ is 3-methoxy-benzyl,R₂ is propyl (particularly i-propyl), R_(2′) is hydrogen; R₁₂ issubstituted imidazoline; R₅, R₆, and R₈ are hydrogen; R₇ is chloro; R₉is methylenedioxyphenyl-; R₁₀, R_(10′), R₁₄, and R_(14′) are hydrogencan be named2-[1-(2-benzo[1,3]dioxol-5-yl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-7-chloro-3-(3-methoxy-benzyl)-chromen-4-one.

Synthesis of the Compounds of Formula I

The compounds of Formula I can be prepared by following the proceduresdescribed with reference to the Reaction Schemes below or utilizingtechniques well known in the art. See, for example, Hirao et al. (1984)Synthesis 1076-1078 and Coppola et al. (1981) Synthesis 523-526, whichare incorporated herein by reference.

Unless specified otherwise, the terms “solvent”, “inert organic solvent”or “inert solvent” mean a solvent inert under the conditions of thereaction being described in conjunction therewith [including, forexample, benzene, toluene, acetonitrile, tetrahydrofuran (“THF”),dimethylformamide (“DMF”), chloroform, methylene chloride (ordichloromethane), diethyl ether, methanol, pyridine and the like].Unless specified to the contrary, the solvents used in the reactions ofthe present invention are inert organic solvents.

The term “q.s.” means adding a quantity sufficient to achieve a statedfunction, e.g., to bring a solution to the desired volume (i.e., 100%).

In general, esters of carboxylic acids may be prepared by conventionalesterification procedures, for example alkyl esters may be prepared bytreating the required carboxylic acid with the appropriate alkanol,generally under acidic conditions. Likewise, amides may be preparedusing conventional amidation procedures, for example amides may beprepared by treating the relevant carboxylic acid with the appropriateamine. Alternatively, a lower-alkyl ester such as a methyl ester of theacid may be treated with an amine to provide the required amide,optionally in presence of trimethylalluminium following the proceduredescribed in Tetrahedron Lett. 48, 4171-4173, (1977 Carboxyl groups maybe protected as alkyl esters, for example methyl esters, which estersmay be prepared and removed using conventional procedures, oneconvenient method for converting carbomethoxy to carboxyl is to useaqueous lithium hydroxide.

The salts and solvates of the compounds mentioned herein may as requiredbe produced by methods conventional in the art. For example, if aninventive compound is an acid, a desired base addition salt can beprepared by treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary, or tertiary); an alkalimetal or alkaline earth metal hydroxide; or the like. Illustrativeexamples of suitable salts include organic salts derived from aminoacids such as glycine and arginine; ammonia; primary, secondary, andtertiary amines; such as ethylenediamine, and cyclic amines, such ascyclohexylamine, piperidine, morpholine, and piperazine; as well asinorganic salts derived from sodium, calcium, potassium, magnesium,manganese, iron, copper, zinc, aluminum, and lithium.

If a compound is a base, a desired acid addition salt may be prepared byany suitable method known in the art, including treatment of the freebase with an inorganic acid, such as hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, and the like, or withan organic acid, such as acetic acid, maleic acid, succinic acid,mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid,glycolic acid, salicylic acid, pyranosidyl acid, such as glucuronic acidor galacturonic acid, alpha-hydroxy acid, such as citric acid ortartaric acid, amino acid, such as aspartic acid or glutamic acid,aromatic acid, such as benzoic acid or cinnamic acid, sulfonic acid,such as p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonicacid, or the like.

Isolation and purification of the compounds and intermediates describedherein can be effected, if desired, by any suitable separation orpurification procedure such as, for example, filtration, extraction,crystallization, column chromatography, thin-layer chromatography orthick-layer chromatography, or a combination of these procedures.Specific illustrations of suitable separation and isolation procedurescan be had by reference to the examples hereinbelow. However, otherequivalent separation or isolation procedures can, of course, also beused.

Brief Description of Reaction Schemes

Reaction Scheme 1 illustrates a synthesis of compounds of formula 109,an intermediate in the synthesis of compounds of Formula I.

Reaction Scheme 2 illustrates a synthesis of compounds of Formula Iwherein R₁₂ is —N(R₄)(COR₃).

Reaction Scheme 3 illustrates a synthesis of compounds of Formula Iwherein R₇ is —OH.

Reaction Scheme 4 illustrates a synthesis of compounds of Formula Iwherein R₇ is —OCH₃.

Reaction Scheme 5 illustrates another synthesis of compounds of FormulaI wherein R₁₂ is —N(R₄)(COR₃).

Reaction Scheme 6 illustrates a synthesis of compounds of Formula Iwherein R₁₂ is —N(R₄)(SO₂R_(3a)).

Reaction Scheme 7 illustrates a synthesis of compounds of Formula Iwherein R₁₂ is —N(R₄)(CH₂R_(3b)).

Reaction Scheme 8 illustrates a synthesis of compounds of Formula Iwherein R₁₂ is optionally substituted imidazolyl.

Reaction Scheme 9 illustrates another synthesis of compounds of FormulaI wherein R₁₂ is optionally substituted imidazolyl.

Reaction Scheme 10 illustrates a synthesis of compounds of Formula Iwherein R₁₂ is optionally substituted imidazolinyl.

Reaction Scheme 11 illustrates a second synthesis of compounds ofFormula I wherein R₁₂ is optionally substituted imidazolinyl.

Reaction Scheme 12 illustrates a synthesis of compounds of Formula Iwherein R₁₂ is —N(R₄)(COR₃) wherein R₃ is —OR₁₅.

Reaction Scheme 13 illustrates a synthesis of compounds of Formula Iwherein R₁₂ is —N(R₄)(COR₃) wherein R₃ is —NHR₁₇.

Reaction Scheme 14 illustrates a synthesis of compounds of Formula 1407which can be used as an intermediate in the synthesis of compounds ofFormula I.

Reaction Scheme 15 illustrates a synthesis of compounds of Formula 1505which can be used as an intermediate in the synthesis of compounds ofFormula I.

Starting Materials

The optionally substituted compounds of Formula 101 are commerciallyavailable, e.g., from Aldrich Chemical Company, Milwaukee, Wis. Otherreactants are likewise commercially available or may be readily preparedby those skilled in the art using commonly employed syntheticmethodology.

Preparation of Compounds of Formula 103

Referring to Reaction Scheme 1, Step 1, about an equivalent of ethylchloroformate is added over about one minute to a 0-5° C. solution of acompound of Formula 101 (preferably wherein the amino protecting group,PG, is a Boc group) and a base such as triethylamine in a nonpolar,aprotic solvent such as THF. After about 15 minutes, a mixture of anexcess of dimethylhydroxylamine hydrochloride (preferably about 1.2equivalents) and a base such as triethylamine in a nonpolar, aproticsolvent such as THF is added over about 5 minutes. The product, acompound of Formula 103, is isolated and used without furtherpurification.

Preparation of Compounds of Formula 105

Referring to Reaction Scheme 1, Step 2, a Grignard reagent is preparedby mixing a compound of formula R₁CH₂Br (generally about 3 equivalents)and magnesium turnings in a nonpolar, aprotic solvent such as diethylether. After about 1.5 hours, the Grignard reaction is generallycomplete. A solution of a compound of Formula 103 in a nonpolar, aproticsolvent such as ether, is added to the Grignard reagent. The temperatureshould be monitored and not allowed to exceed ˜30° C. The product, acompound of Formula 105, is isolated and purified.

Preparation of Compounds of Formula 107

Referring to Reaction Scheme 1, Step 3, lithium bis(trimethylsilyl)amide(about 3.3 equivalents) is added slowly over ˜3 minutes to a −78° C.solution of a compound of Formula 105 in a nonpolar, aprotic solventsuch as THF. The reaction solution temperature should be monitored andthe addition of base conducted at a rate sufficient to prevent thetemperature from exceeding about −54° C. After the addition is complete,the resulting solution is maintained at −78° C. for about 30 minutes. Anacid chloride of Formula 106 (preferably, neat) is then added. Thereaction solution is maintained at −78° C. for about 30 minutes. Theproduct is isolated and used without further purification.

A mixture of the above crude product, a base such as potassiumcarbonate, and a polar, aprotic solvent such as DMF is maintained atabout room temperature for about 30 minutes. The product, a compound ofFormula 107 is isolated and purified.

Preparation of Compounds of Formula 109

Referring to Reaction Scheme 1, Step 4, optionally, the protectinggroup, PG, may be removed from the amine. One of skill in the art willappreciate that the conditions for removal of the protecting group willvary with different protecting groups. Such conditions are well known inthe art and can be found, e.g., in Greene et al. supra. When PG is Boc,it may be removed by treatment of a compound of Formula 107 with amixture of aqueous TFA (preferably TFA:H₂O, 97.5:2.5) at roomtemperature. The product, a compound of Formula 109, is isolated andpurified.

Preparation of Optically Active Compounds

In compounds of the invention where R₂ differs from R_(2′), a particularstereo configuration (such as the (R) isomer) may be preferred at thestereogenic center to which R₂ and R_(2′) are attached. The opticallyactive compound can be prepared by methods known in the art. Forexample, an amine of Formula 109 is dissolved in an inert organicsolvent (such as IPA) and warmed to 60° C. In a separate vessel, aresolving agent (such as dibenzoyl-D-tartaric acid) is dissolved,preferably in the same warm solvent, and then quickly added (withagitation) to the warm amine solution. The reaction mixture is left tocrystallize by cooling to room temperature over 16 hours undercontinuing agitation. The desired isomer, e.g., the (R) isomer of acompound of Formula 109, is isolated and purified.

For the sake of brevity in the remaining description of the synthesis ofcompounds of Formula I, it should be understood that either singleisomer or a mixture of isomers may be employed to give the correspondingproduct.

Preparation of Formula 203

Referring to Reaction Scheme 2, Step 1, to a solution of a compound ofFormula 109 is added successively a slight excess (preferably about 1.2equivalents) of an aldehyde comprising R_(4′) (i.e., a compound havingthe formula R_(4′)CHO where R_(4′)CH₂— is equivalent to R₄ and R₄ is asdescribed above or is a protected precursor to such a substituent, e.g.,(3-oxo-propyl)-carbamic acid tert-butyl ester) and a reducing agent suchas sodium triacetoxyborohydride. The resulting mixture is stirred forseveral hours. The product, a compound of Formula 203 is isolated andpurified.

Preparation of Formula 205

Referring to Reaction Scheme 2, Step 2, to a solution of a compound ofFormula 203 and an amine base such as diisopropylethylamine in anonpolar, aprotic solvent such as dichloromethane is added an R₃ acylchloride (such as Cl—C(O)—R₃ where R₃ is as described above). Theresulting solution is stirred under nitrogen at room temperature forseveral hours. The product, a compound of Formula 205 is isolated andpurified.

Preparation of Formula 207

Optionally, any protecting groups on compounds of Formula 205 are thenremoved. For example, if R₄ comprises a protected amine wherein theprotecting group is a Boc group, the Boc group can be removed bytreatment of the compound of Formula 205 with an acid such astrifluoroacetic acid in a nonpolar, aprotic solvent such asdichloromethane, while maintaining the reaction at about roomtemperature. The reaction is monitored e.g., by TLC. Upon completion,the product, a compound of Formula 207 is isolated and purified.

Preparation of Compounds of Formula 303

Referring to Reaction Scheme 3, Step 1, to a solution of a compound ofFormula 301 in a nonpolar, aprotic solvent such as DMF is added sodiumhydride. The resulting solution was stirred at about 45° C. for about 5minutes, then allyl alcohol (about 1.4 equivalents) is added viapipette. The resulting solution is stirred at about 45° C. for about 12hours and then cooled to room temperature. The product, a compound ofFormula 303, is isolated and used without further purification.

Preparation of Compounds of Formula 305

Referring to Reaction Scheme 3, Step 2, to a room temperature solutionof a compound of Formula 303, in an aprotic solvent such asacetonitrile, is added morpholine followed by Pd(PPh₃)₄. The resultingsolution is stirred for about 5 minutes. The product, a compound ofFormula 305, is isolated and purified.

Referring to Reaction Scheme 4, a compound of Formula 401 is dissolvedin 0.5 M sodium methoxide in methanol and heated to about 70° C. Thetemperature is maintained at about 70° C. for about 12 hours and thencooled to room temperature. The product, a compound of Formula 403, isisolated and purified.

Preparation of Compounds of Formula 503

Referring to Reaction Scheme 5, Step 1, to a solution of a compound ofFormula 501 and a base such as triethylamine in a nonpolar, aproticsolvent such as CH₂Cl₂ at room temperature is added about an equivalentof an acid chloride of formula Cl—(CO)—CH₂R₁ over about 5 minutes. Afterabout 30 minutes, the product, a compound of Formula 503, is isolatedand used without any further purification.

Preparation of Compounds of Formula 505

Referring to Reaction Scheme 5, Step 2, AlCl₃ (about 1.3 equivalents) isadded slowly over about 15 minutes to a compound of Formula 503 at about140° C. After gas evolution has ceased, the reaction mixture is cooledto room temperature. The product, a compound of Formula 505, is isolatedand purified.

Preparation of Compounds of Formula 507

Referring to Reaction Scheme 5, Step 3, a solution of a compound ofFormula 505 and an amino acid of Formula 506 (preferably about 1.1equivalents) in which the amine group has been suitably protected with aprotecting group PG (preferably a Boc group), a coupling reagent such asHBTU (preferably about 1.2 equivalents), a base such as TEA, and anonpolar, aprotic solvent such as CH₂Cl₂ is maintained at roomtemperature for about 5 hours. The product, a compound of Formula 507,is isolated and purified.

Preparation of Compounds of Formula 509

Referring to Reaction Scheme 5, Step 4, a mixture of ester of Formula507 and a base such as potassium carbonate in a polar, aprotic solventsuch as DMF is heated at about 140° C. After about 30 mins, the product,a compound of Formula 509, is isolated and purified.

Preparation of Compounds of Formula 511

Referring to Reaction Scheme 5, Step 5, the amine protecting group PG isthen removed. When PG is Boc, this may be accomplished by treatment of acompound of formula 509 with aqueous acid (preferably, 97.5:2.5 TFA:H₂O)at room temperature for about one hour. The free amine is isolated andused without further purification.

The resulting product; an aldehyde comprising R_(4′) (i.e., a compoundhaving the formula R_(4′)CHO where R_(4′)CH₂— is equivalent to R₄ and R₄is as described above or is a protected precursor to such a substituent,e.g., (3-oxo-propyl)-carbamic acid tert-butyl ester; preferably, about1.45 equivalents); a reducing reagent, such as Na(OAc)₃BH; and anonpolar, aprotic solvent such as CH₂Cl₂ is maintained at roomtemperature for about 3 hours. The product, a compound of Formula 511,is isolated and purified.

Preparation of Compounds of Formula 513

Referring to Reaction Scheme 5, Step 6, to a solution of a compound ofFormula 511, a base such as diisoproylethylamine, and a nonpolar,aprotic solvent such as CH₂Cl₂ at room temperature is added an acidchloride of the formula R₃COCl (preferably about 2 equivalents). Afterabout 2.5 h, the product, a compound of Formula 513, is isolated andpurified.

Referring to Reaction Scheme 6, to a solution of a compound of Formula203 and an amine base such as diisopropylethylamine in a nonpolar,aprotic solvent such as dichloromethane is added a compound having theformula Cl—S(O)₂—R_(3a) or O—(S(O)₂—R_(3a))₂ where R_(3a) is asdescribed above. The resulting solution is stirred under nitrogen atroom temperature for several hours. The product, a compound of Formula603 is isolated and purified.

Referring to Reaction Scheme 7, to a solution of a compound of Formula203 and an amine base such as diisopropylethylamine in a nonpolar,aprotic solvent such as dichloromethane is added a compound having theformula X—CH₂—R_(3b) where R_(3b) is as described above and X is Br, Cl,mesylate, or tosylate. The resulting solution is stirred under nitrogenat room temperature or with heat for several hours. The product, acompound of Formula 703 is isolated and purified.

Preparation of Formula 803

Referring to Reaction Scheme 8, Step 1, to an optionally substitutedcompound of Formula 109 dissolved in a polar, aprotic solvent (such asDMF) in the presence of a base (such as potassium carbonate) is addedone equivalent of an optionally substituted suitably protected aldehydewherein such aldehyde further comprises a leaving group, preferably, ahalide (such as bromoacetaldehyde dimethylacetal). The solution isheated at reflux, monitoring completion of the reaction (e.g., by TLC).The reaction mixture is cooled and the corresponding, optionallysubstituted compound of Formula 803 is isolated and purified.

Preparation of Formula 805

Referring to Reaction Scheme 8, Step 2, to an optionally substitutedcompound of Formula 803 in an inert solvent (such as dichloromethane) inthe presence of about 1.5 molar equivalents of an amine base (such astriethylamine) is added about 1.5 molar equivalents of an R₉ acidchloride, such as, Cl—C(O)—R₉, where R₉ is as described herein. Thereaction takes place, with stirring, at room temperature over a periodof 4 to 24 hours. Completion is monitored, e.g., by TLC. Thecorresponding compound of Formula 805 is isolated and purified.

Preparation of Formula 807

Referring to Reaction Scheme 8, Step 3, a solution of a compound ofFormula 805 and an excess of ammonium acetate in acetic acid is heatedat reflux for 1-4 hours. Completion is monitored, e.g., by TLC. Thecorresponding compound of Formula 807 is isolated and purified.

Preparation of Formula 903

Referring to Reaction Scheme 9, Step 1, a suspension of a compound ofFormula 109, an alpha-haloketone reagent of the Formula R_(13′)(CO)CH₂Xwherein X is a halide and R_(13′) is as described herein, and about anequivalent of a base, such as potassium carbonate in a polar, aproticsolvent such as DMF is stirred at room temperature. The reaction isdiluted with water and the resulting compound, a compound of Formula903, typically a sold, is used in the subsequent step withoutpurification. Where the resulting compound is not a solid, it isisolated using standard procedures and then used in the subsequent step.

Preparation of Formula 905

Referring to Reaction Scheme 9, Step 2, a solution of the compound ofFormula 903, about an equivalent of an amine base, such as triethylamineand about an equivalent of an acid chloride (such as a compound ofFormula R₉—COCl) in an organic solvent such as methylene chloride isstirred at room temperature for several hours. Completion is monitored,e.g., by TLC. The corresponding compound of Formula 905 is isolated andpurified.

Preparation of Formula 907

Referring to Reaction Scheme 9, Step 3, a solution of a compound ofFormula 905 and an excess of ammonium acetate in acetic acid is heatedat reflux using a Dean-Stark trap and condenser. Completion ismonitored, e.g., by TLC. The corresponding compound of Formula 907 isisolated and purified.

Preparation of Formula 909

Referring to Reaction Scheme 9, Step 4, when R_(13′) comprises aprotected aminoalkyl group, the amino protected group may be removed.For example, when the amino group is protected as the correspondingphthalimide, the protecting group is removed as follows. A solution of acompound of Formula 907 and an excess of anhydrous hydrazine in a polar,protic solvent such as ethanol is heated at reflux. The reaction iscooled to about 5° C. and any precipitate is filtered off. The filtrateis concentrated in vacuo and purified to yield a compound of Formula909. One of skill in the art will appreciate that other conditions maybe used to remove other protecting groups.

Preparation of Formula 1003

Referring to Reaction Scheme 10, Step 1, reductive amination of aminesof Formula 109 with an optionally substituted, aldehyde-containingcarbamic acid ester gives urethane intermediates. More specifically, toa solution of a compound of Formula 109 and an equivalent of a suitablyprotected aldehyde (Seki et. al. Chem. Pharm. Bull. 1996, 44, 2061) indichloromethane is added a slight excess of a reducing agent, such assodium triacetoxyborohydride. The resultant cloudy mixture is maintainedat ambient temperature. Completion is monitored, e.g., by TLC. Thecorresponding compound of Formula 1003 is isolated and used in thesubsequent step without purification.

Preparation of Formula 1005

Referring to Reaction Scheme 10, Step 2, the amino protecting group PGis then removed. When PG is a Boc protecting group, this may beaccomplished by the treatment of a solution of a compound of Formula1003 in a nonpolar, aprotic solvent such as dichloromethane with astrong acid such as trifluoroacetic acid. The resultant solution ismaintained at ambient temperature overnight and concentrated underreduced pressure. The residue is isolated to give a compound of Formula1005 which was used in the subsequent step without purification.

Preparation of Formula 1007

Referring to Reaction Scheme 10, Step 3, to a solution of a compound ofFormula 1005 in a nonpolar, aprotic solvent such as dichloromethane isadded an excess, preferably about two equivalents of an amine base suchas triethylamine, followed by about an equivalent or slight excess of anacid chloride of the formula R₉—CO—Cl. The resultant solution is stirredat ambient temperature for about 3 hours. Completion is monitored, e.g.,by TLC. The corresponding compound of Formula 1007 is isolated andpurified.

Preparation of Formula 1009

Referring to Reaction Scheme 10, Step 4, a solution of a compound ofFormula 1007 in an excess of phosphorus oxychloride is heated at reflux.After 8 hours, the reaction mixture is allowed to cool to ambienttemperature and concentrated under reduced pressure. The correspondingcompound of Formula 1009 is isolated and purified.

Preparation of Formula 1109

As an alternative to Steps 3 and 4 of Reaction Scheme 10, acylation ofprimary amines of Formula 1005, followed by acetic acid mediatedcyclization, can proceed without isolation of the intermediate amides toprovide the target compound of Formula 1109. This route is shown inReaction Scheme 11.

More specifically, to a solution of a compound of Formula 1005 in anonpolar, aprotic solvent such as dichloromethane is added an excess,preferably about two equivalents of an amine base, such astriethylamine, followed by about an equivalent of an acid chloride ofthe formula R₉—CO—Cl. The resultant solution is stirred at ambienttemperature for 2 hours, then evaporated under reduced pressure. Theresultant solid is treated with glacial acetic acid, then the resultantsuspension is heated at reflux for about 48 hours. The reaction iscooled to ambient temperature then evaporated under reduced pressure.The corresponding compound of Formula 1109 is isolated and purified.

Referring to Reaction Scheme 12, a compound of Formula 203 is reactedwith a slight excess of a compound of the formula R₁₅O(CO)Cl in thepresence of a base such as triethylamine in a nonpolar, aprotic solventsuch as dichloromethane. The product, a compound of Formula 1203 isisolated and purified.

Referring to Reaction Scheme 13, a compound of Formula 203 is treatedwith a slight excess of an isocyanate R₁₇—N═C═O in the presence of abase, such as triethylamine, in a nonpolar, aprotic solvent, such asdichloromethane. The product, a compound of Formula 1303, is isolatedand purified.

Preparation of Compounds of Formula 1403

Referring to Reaction Scheme 14, Step 1, a nonpolar, aprotic solvent,such as THF, and an excess of a solution of an optionally substitutedvinyl magnesium bromide in a nonpolar, aprotic solvent (and morepreferably, about three equivalents of a 1.0 M solution of an optionallysubstituted vinyl magnesium bromide in THF) is cooled to −78° C. whilestirring under a nitrogen atmosphere. The mixture is treated dropwisewith a solution of a compound of Formula 1401 in a nonpolar, aproticsolvent, such as THF over about 30 min. After the mixture is stirred for30 min at −78° C., the cooling bath is removed and the reaction mixtureis warmed slowly to room temperature overnight (about 15 h). Theproduct, a compound of Formula 1403, is isolated and purified.

Preparation of Compounds of Formula 1405

Referring to Reaction Scheme 14, Step 2, to a solution of a compound ofFormula 1403 in an anhydrous, nonpolar, aprotic solvent, such asacetonitrile under an inert atmosphere, such as argon, is added about anequivalent of a compound of the Formula R_(1′)—X wherein R_(1′) is anoptionally substituted vinyl, optionally substituted aryl, or optionallysubstituted heteroaryl and X is I, Br, or —OTf, and a base such astriethylamine followed by palladium (II) acetate (preferably, about0.025 equivalents). The resulting solution is heated to about 80° C.After about 15 h, the reaction mixture is allowed to cool to roomtemperature. The product, a compound of Formula 1405, is isolated andimmediately purified.

Preparation of Compounds of Formula 1407

To a solution of a compound of Formula 1405 in a nonpolar, aproticsolvent such as ethyl acetate under nitrogen is added 10 wt % palladiumon carbon. The nitrogen is replaced with a balloon of hydrogen and theflask is purged. After 3 h, the reaction flask is purged with nitrogenand filtered through a pad of celite (rinsing with a solvent such asethyl acetate). The product, a compound of Formula 1407 is isolated andpurified.

Preparation of Compounds of Formula 1503

Referring to Reaction Scheme 15, Step 1, about one equivalent ofcarbonyldiimidazole is added slowly to a room temperature solution of acompound of Formula 1501 (preferably wherein the amino protecting groupPG is Boc) in a nonpolar, aprotic solvent such THF. After about onehour, the product, a compound of Formula 1503, is isolated and usedwithout further purification.

Preparation of Compounds of Formula 1505

Referring to Reaction Scheme 15, Step 2, a Grignard reagent is preparedfrom a compound of Formula R₁CH₂Br and magnesium turnings in a nonpolar,aprotic solvent such as THF. A solution of a compound of Formula 1503 ina nonpolar, aprotic solvent such as THF is cooled to about 0-5° C. Thesolution of the Grignard reagent is then added via syringe to the 0-5°C. solution of the compound of Formula 1503. The temperature ismonitored by internal thermometer and is not allowed to exceed about 15°C. The reaction mixture is maintained at about 0-5° C. for about onehour. The product, a compound of Formula 1505, is isolated and purified.

Preferred Processes and Last Steps

A compound of Formula I is optionally contacted with a pharmaceuticallyacceptable acid or base to form the corresponding acid or base additionsalt.

A pharmaceutically acceptable acid addition salt of a compound ofFormula I is optionally contacted with a base to form the correspondingfree base of Formula I. A pharmaceutically acceptable base addition saltof a compound of Formula I is optionally contacted with an acid to formthe corresponding free acid of Formula I.

Preferred Compounds

Preferred R₁ when Either R₂ or R_(2′) is not Hydrogen

When considering the compounds of Formula I, in a preferred embodimentwhen either one or both R₂ or R_(2′) is not hydrogen (more preferably,either one of R₂ or R_(2′) is not hydrogen), R₁ is selected fromhydrogen, optionally substituted C₁-C₈ alkyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedaryl-C₁-C₄-alkyl-, and optionally substituted heteroaryl-C₁-C₄-alkyl-(more preferably optionally substituted aryl and optionally substitutedaryl-C₁-C₄-alkyl-). In a more preferred embodiment R₁ is selected fromhydrogen, optionally substituted C₁-C₄ alkyl, optionally substitutedphenyl-C₁-C₄-alkyl-, optionally substituted naphthalenylmethyl,optionally substituted phenyl, and naphthyl. More preferably, R₁ isoptionally substituted phenyl-C₁-C₄-alkyl- or optionally substitutedheteroaryl-C₁-C₄-alkyl-.

In a most preferred embodiment R₁ is naphthyl, phenyl, bromophenyl,chlorophenyl, methoxyphenyl, ethoxyphenyl, tolyl, dimethylphenyl,chorofluorophenyl, methylchlorophenyl, ethylphenyl, phenethyl, benzyl,chlorobenzyl, methylbenzyl, methoxybenzyl, cyanobenzyl, hydroxybenzyl,dichlorobenzyl, dimethoxybenzyl, or naphthalenylmethyl. More preferably,R₁ is benzyl, cyanobenzyl, methoxybenzyl, or naphthalenylmethyl. Mostpreferably, R₁ is benzyl.

Preferred R₁ when R₂ and R_(2′) are Hydrogen

In an embodiment wherein R₂ and R_(2′) are both hydrogen, preferably R₁is chosen from optionally substituted aryl, optionally substitutedaryl-C₁-C₄-alkyl-, optionally substituted heteroaryl, and optionallysubstituted heteroaryl-C₁-C₄-alkyl-, provided however, that R₁ is notsubstituted phenyl. More preferably, R₁ is optionally substitutedaryl-C₁-C₄-alkyl- or optionally substituted heteroaryl-C₁-C₄-alkyl-.More preferably, when R₂ and R_(2′) are both hydrogen, R₁ is selectedfrom optionally substituted phenyl-C₁-C₄-alkyl, and optionallysubstituted naphthalenylmethyl. In a more preferred embodiment whereinR₂ and R_(2′) are both hydrogen, R₁ is chosen from benzyl, chlorobenzyl,methylbenzyl, methoxybenzyl, cyanobenzyl, hydroxybenzyl, dichlorobenzyl,dimethoxybenzyl, and naphthalenylmethyl. More preferably, R₁ is benzyl,cyanobenzyl, methoxybenzyl, or naphthalenylmethyl. More preferably, R₁is benzyl.

Preferred R₂

When considering the compounds of Formula I and as will be appreciatedby those skilled in the art, the compounds described herein possess apotentially chiral center at the carbon to which R₂ and R_(2′) areattached. The R₂ and R_(2′) groups may be the same or different; ifdifferent, the compound is chiral (i.e., has a stereogenic center). WhenR₂ and R_(2′) are different, in preferred embodiments R_(2′) is hydrogenand R₂ is other than hydrogen. The invention contemplates the use ofpure enantiomers and mixtures of enantiomers, including racemicmixtures, although the use of a substantially optically pure enantiomerwill generally be preferred. The term “substantially optically pure” or“enantiomerically pure” means having at least about 95% of the describedenantiomer with no single impurity greater than about 1% and preferably,at least about 97.5% enantiomeric excess. In a preferred embodiment, thestereogenic center to which R₂ and R_(2′) are attached is of the Rconfiguration.

In one embodiment, R₂ is optionally substituted C₁-C₄ alkyl, and R_(2′)is hydrogen or optionally substituted C₁-C₄ alkyl. More preferably,R_(2′) is hydrogen and R₂ is optionally substituted C₁-C₄ alkyl. In amost preferred embodiment R₂ is chosen from methyl, ethyl, propyl(particularly, c-propyl or i-propyl), butyl (particularly, t-butyl),methylthioethyl, methylthiomethyl, aminobutyl, (CBZ)aminobutyl,cyclohexylmethyl, benzyloxymethyl, methylsulfinylethyl,methylsulfinylmethyl, and hydroxymethyl, and R_(2′) is hydrogen.Especially preferred is when R_(2′) is hydrogen and R₂ is ethyl orpropyl (particularly, c-propyl or i-propyl). More preferably, R₂ isi-propyl. More preferred is the embodiment wherein the stereogeniccenter to which R₂ and R_(2′) is attached is of the R configuration.

In another embodiment, both R₂ and R_(2′) are hydrogen.

Preferred R₃ Groups when R₁₂ is —N(R₄)(COR₃)

When considering the compounds of Formula I wherein R₁₂ is —N(R₄)(COR₃),in a preferred embodiment R₃ is selected from optionally substitutedC₁-C₈ alkyl, optionally substituted aryl-C₁-C₄-alkyl-, optionallysubstituted heteroaryl-C₁-C₄-alkyl-, optionally substituted heteroaryl,optionally substituted aryl, R₁₅O— and R₁₇—NH—, R₁₅ is chosen fromoptionally substituted C₁-C₈ alkyl and optionally substituted aryl, andR₁₇ is chosen from hydrogen, optionally substituted C₁-C₈ alkyl andoptionally substituted aryl. Preferred R₃ are optionally substitutedC₁-C₈ alkyl (e.g., C₁-C₈ alkyl substituted with lower-alkoxy),optionally substituted heteroaryl, and optionally substituted aryl.

In a more preferred embodiment, when R₃ is not R₁₇NH— or R₁₅O—, R₃ ischosen from phenyl; phenyl substituted with one or more of the followingsubstituents: halo, C₁-C₄ alkyl, C₁-C₄ alkyl substituted with hydroxy(e.g., hydroxymethyl), C₁-C₄ alkoxy, C₁-C₄ alkyl substituted with C₁-C₄alkoxy, nitro, formyl, carboxy, cyano, methylenedioxy, ethylenedioxy,acyl (e.g., acetyl), —N-acyl (e.g., N-acetyl) or trifluoromethyl;benzyl; phenoxymethyl-; halophenoxymethyl-; phenylvinyl-; heteroaryl-;heteroaryl- substituted with C₁-C₄ alkyl or C₁-C₄ alkyl substituted withhalo (e.g., CF₃); C₁-C₄ alkyl substituted with C₁-C₄ alkoxy- andbenzyloxymethyl-.

In a most preferred embodiment, when R₃ is not R₁₇NH— or R₁₅O—, R₃ ischosen from phenyl, halophenyl, dihalophenyl, cyanophenyl,halo(trifluoromethyl)phenyl, hydroxymethylphenyl, methoxyphenyl,ethoxyphenyl, carboxyphenyl, ethylphenyl, tolyl, methylenedioxyphenyl,ethlenedixoyphenyl, methoxychlorophenyl, dihydro-benzodioxinyl,methylhalophenyl, trifluoromethylphenyl,bis(trifluoromethyl)phenylbenzyl, furanyl, C₁-C₄ alkyl substitutedfuranyl, trifluoromethylfuranyl, C₁-C₄ alkyl substitutedtrifluoromethylfuranyl, benzofuranyl, thiophenyl, C₁-C₄ alkylsubstituted thiophenyl, benzothiophenyl, benzothiadiazolyl, pyridinyl,indolyl, methylpyridinyl, trifluoromethylpyridinyl, pyrrolyl,quinolinyl, picolinyl, pyrazolyl, C₁-C₄ alkyl substituted pyrazolyl,N-methylpyrazolyl, C₁-C₄ alkyl substituted N-methylpyrazolyl, C₁-C₄alkyl substituted pyrazinyl, C₁-C₄ alkyl substituted isoxazolyl,benzoisoxazolyl, morpholinomethyl, methylthiomethyl, methoxymethyl,N-methyl imidazolyl, and imidazolyl. Yet more preferably, R₃ is tolyl,halophenyl, halomethylphenyl, hydroxymethylphenyl, methylenedioxyphenyl,formylphenyl or cyanophenyl.

In a more preferred embodiment, when R₃ is R₁₇NH—, R₁₇ is chosen fromhydrogen, C₁-C₄ alkyl; cyclohexyl; phenyl; and phenyl substituted withhalo, C₁-C₄ alkyl, C₁-C₄ alkoxy, or C₁-C₄ alkylthio.

In a most preferred embodiment, when R₃ is R₁₇NH—, R₁₇ is hydrogenisopropyl, butyl, cyclohexyl, phenyl, bromophenyl, dichlorophenyl,methoxyphenyl, ethylphenyl, tolyl, trifluoromethylphenyl ormethylthiophenyl.

In an embodiment, wherein R₃ is R₁₅O—, R₁₅ is chosen from optionallysubstituted C₁-C₈ alkyl and optionally substituted aryl.

Preferred R_(3a) Groups when R₁₂ is —N(R₄)(SO₂R_(3a))

Preferably, when R₁₂ is —N(R₄)(SO₂R_(3a)), R_(3a) is chosen from C₁-C₁₃alkyl; phenyl; naphthyl; phenyl substituted with halo, C₁-C₄ alkyl,C₁-C₄ alkoxy, cyano, nitro, methylenedioxy, or trifluoromethyl;biphenylyl and heteroaryl. More preferably, R_(3a) is chosen from phenylsubstituted with halo, C₁-C₄ alkyl, C₁-C₄ alkoxy, cyano, nitro,methylenedioxy, or trifluoromethyl and naphthyl.

Preferred R_(3b) Groups when R₁₂ is —N(R₄)(CH₂R_(3b))

Preferably, when R₁₂ is —N(R₄)(CH₂R_(3b)), R_(3b) is chosen from C₁-C₁₃alkyl; substituted C₁-C₄ alkyl; phenyl; naphthyl; phenyl substitutedwith carboxy, alkoxycarbonyl cyano, halo, C₁-C₄ alkyl-, C₁-C₄ alkoxy,nitro, methylenedioxy, or trifluoromethyl; biphenylyl, benzyl; andheterocyclyl.

Most preferably, R_(3b) is chosen from halophenyl, polyhalophenyl,methylhalophenyl, tolyl, dimethylphenyl, methoxyphenyl, dimethoxyphenyl,cyanophenyl, trifluoromethylphenyl, trifluoromethoxyphenyl,bis(trifluoromethyl)phenyl, carboxyphenyl, t-butylphenyl,methoxycarbonylphenyl, piperidinyl and naphthyl.

Preferred R₄ Groups when R₁₂ is —NHR₄, —N(R₄)(COR₃), or—N(R₄)(CH₂R_(3b))

In a preferred embodiment when R₁₂ is —NHR₄, —N(R₄)(COR₃), or—N(R₄)(CH₂R_(3b)), R₄ is chosen from hydrogen, optionally substitutedC₁-C₁₃ alkyl, optionally substituted aryl, optionally substitutedaryl-C₁-C₄-alkyl-, optionally substituted heterocyclyl, and optionallysubstituted heteroaryl-C₁-C₄-alkyl- (preferably hydrogen or optionallysubstituted C₁-C₁₃ alkyl).

More preferably, R₄ is chosen from hydrogen, C₁-C₄ alkyl; cyclohexyl;phenyl substituted with hydroxyl, C₁-C₄ alkoxy or C₁-C₄ alkyl; benzyl;heteroarylmethyl-; heteroarylethyl-; heteroarylpropyl-; andR₁₆-alkylene-, wherein R₁₆ is hydroxyl, di(C₁-C₄ alkyl)amino-, (C₁-C₄alkyl)amino-, amino, C₁-C₄ alkoxy-, or N-heterocyclyl-, particularlypyrrolidino, piperidino or imidazolyl.

More preferably, R₄ is R₁₆-alkylene-, wherein R₁₆ is amino, C₁-C₄alkylamino-, di(C₁-C₄ alkyl)amino-, C₁-C₄ alkoxy-, hydroxyl, orN-heterocyclyl. Preferably R₁₆ is amino.

In a most preferred embodiment when R₁₂ is —NHR₄, —N(R₄)(COR₃), or—N(R₄)(CH₂R_(3b)), R₄ is chosen from hydrogen, methyl, ethyl, propyl,butyl, cyclohexyl, carboxyethyl, carboxymethyl, methoxyethyl,hydroxyethyl, hydroxypropyl, dimethylaminoethyl, dimethylaminopropyl,diethylaminoethyl, diethylaminopropyl, aminopropyl, methylaminopropyl,2,2-dimethyl-3-(dimethylamino)propyl,1-cyclohexyl-4-(diethylamino)butyl, aminoethyl, aminobutyl, aminopentyl,aminohexyl, aminoethoxyethyl, isopropylaminopropyl,diisopropylaminoethyl, 1-methyl-4-(diethylamino)butyl,(t-Boc)aminopropyl, hydroxyphenyl, benzyl, methoxyphenyl,methylmethoxyphenyl, dimethylphenyl, tolyl, ethylphenyl,(oxopyrrolidinyl)propyl, (methoxycarbonyl)ethyl, benzylpiperidinyl,pyridinylethyl, pyridinylmethyl, morpholinylethyl morpholinylpropyl,piperidinyl, azetidinylmethyl, azetidinylethyl, azetidinylpropylpyrrolidinylethyl, pyrrolidinylpropyl, piperidinylmethyl,piperidinylethyl, imidazolylpropyl, imidazolylethyl,(ethylpyrrolidinyl)methyl, (methylpyrrolidinyl)ethyl,(methylpiperidinyl)propyl, (methylpiperazinyl)propyl, furanylmethyl andindolylethyl.

More preferably, R₄ is aminoethyl, aminopropyl, aminobutyl, aminopentyl,aminohexyl, methylaminoethyl, methylaminopropyl, methylaminobutyl,methylaminopentyl, methylaminohexyl, dimethylaminoethyl,dimethylaminopropyl, dimethylaminobutyyl, dimethylaminopentyl,dimethylaminohexyl, ethylaminoethyl, ethylaminopropyl, ethylaminobutyl,ethylaminopentyl, ethylaminohexyl, diethylaminoethyl,diethylaminopropyl, diethylaminobutyyl, diethylaminopentyl, ordiethylaminohexyl, most preferably aminopropyl.

Preferred R₄ Groups when R₁₂ is —N(R₄)(SO₂R_(3a))

Preferably, when R₁₂ is —N(R₄)(SO₂R_(3a)), R₄ is chosen from C₁-C₄alkyl, cyclohexyl; phenyl substituted with hydroxyl, C₁-C₄ alkoxy orC₁-C₄ alkyl; benzyl; heteroarylmethyl-; heteroarylethyl-;heteroarylpropyl-; heteroarylethyl-; heteroarylpropyl- andR₁₆-alkylene-, wherein R₁₆ is hydroxyl, di(C₁-C₄ alkyl)amino-, (C₁-C₄alkyl)amino-, amino, C₁-C₄ alkoxy-, or N-heterocyclyl-, particularlypyrrolidino, piperidino or imidazolyl.

R₁₂ is an Imidazole

Preferably, when R₁₂ is an imidazole, R₁₂ has the formula:

wherein

R₉ is chosen from hydrogen, optionally substituted C₁-C₈ alkyl,optionally substituted aryl, optionally substituted aryl-C₁-C₄-alkyl-,optionally substituted heteroaryl-C₁-C₄-alkyl-, optionally substitutedaryl-C₁-C₄-alkoxy-, optionally substituted heteroaryl-C₁-C₄-alkoxy-,optionally substituted heteroaryl-; and R₁₃ and R_(13′) areindependently hydrogen, optionally substituted C₁-C₈ alkyl, optionallysubstituted aryl, or optionally substituted aryl-C₁-C₄-alkyl-(preferably optionally substituted aryl). More preferably, R₉ is phenylsubstituted with C₁-C₄-alkyl, C₁-C₄-alkoxy-, and/or halo (especiallyC₁-C₄-alkyl and/or halo); phenyl; or benzyl. Yet more preferably, R₉ istolyl; halophenyl; or halomethylphenyl.

In a preferred embodiment, R₁₃ is hydrogen and R_(13′) is substitutedC₁-C₄ alkyl. More preferably, R₁₃ is hydrogen and R_(13′) isaminomethyl, aminoethyl, aminopropyl, acetylamino-methyl,acetylaminoethyl, benzyloxycarbonylamino-methyl orbenzyloxycarbonylamino-ethyl.

R₁₂ is an Imidazoline

Preferably, when R₁₂ is an imidazoline, R₁₂ has the formula

wherein R₉ is chosen from hydrogen, optionally substituted C₁-C₈ alkyl,optionally substituted aryl, optionally substituted aryl-C₁-C₄-alkyl-,and optionally substituted heteroaryl-; and R₁₀, R_(10′), R₁₄, andR_(14′) are independently chosen from hydrogen, optionally substitutedC₁-C₈ alkyl, optionally substituted aryl, and optionally substitutedaryl-C₁-C₄-alkyl-. More preferably, R₉ is methylenedioxyphenyl; phenyl;phenyl substituted with C₁-C₄ alkyl, C₁-C₄ alkoxy, and/or halo; orbenzyl. In a preferred embodiment, R₉ is methylenedioxyphenyl-; phenyl;or phenyl substituted with methoxy, halo and/or methyl (preferably haloand/or methyl, including tolyl), more preferably methylenedioxyphenyl orsaid substituted phenyls. In another preferred embodiment, R₁₀, R_(10′),R_(14′), and R₁₄ are independently hydrogen or optionally substitutedalkyl (preferably optionally substituted C₁-C₄ alkyl). More preferably,R₁₀ and R_(10′) are independently selected from the group consisting ofhydrogen or optionally substituted C₁-C₄ alkyl (and more particularly,methyl or aminoalkyl-) and R_(14′) and R₁₄ are hydrogen.Preferred R₅, R₆, R₇, and R₈ Groups

When considering the compounds of Formula I, in other preferredembodiments R₅, R₆, R₇ and R₈ are independently chosen from hydrogen;acyl, alkyl; alkyl substituted with alkyl, alkoxy, halo, hydroxyl,nitro, cyano, dialkylamino, alkylsulfonyl, alkylsulfonamido, alkylthio,carboxyalkyl, carboxamido, aminocarbonyl, lower-alkylaminocarbonyl-(e.g. methylaminocarbonyl- or ethylaminocarbonyl-),di(lower-alkyl)aminocarbonyl- (e.g. dimethylaminocarbonyl- ordiethylaminocarbonyl-), aryl, or heteroaryl; alkoxy; alkoxy substitutedwith alkyl, acyl, alkoxy, halo, hydroxyl, nitro, cyano, dialkylamino,alkylsulfonyl, alkylsulfonamido, alkylthio, carboxyalkyl, carboxamido,aminocarbonyl, lower-alkylaminocarbonyl- (e.g. methylaminocarbonyl- orethylaminocarbonyl-), di(lower-alkyl)aminocarbonyl- (e.g.dimethylaminocarbonyl- or diethylaminocarbonyl-), aryl, or heteroaryl;halogen; hydroxyl; nitro; cyano; dialkylamino; alkylsulfonyl;alkylsulfonamido; alkylthio; carboxyalkyl; carboxamido; amidocarbonyl;aryl; aryl substituted with alkyl, acyl, alkoxy, halo, hydroxyl, nitro,cyano, dialkylamino, alkylsulfonyl, alkylsulfonamido, alkylthio,carboxyalkyl, carboxamido, aminocarbonyl, lower-alkylaminocarbonyl-(e.g. methylaminocarbonyl- or ethylaminocarbonyl-),di(lower-alkyl)aminocarbonyl- (e.g. dimethylaminocarbonyl- ordiethylaminocarbonyl-), aryl, or heteroaryl; heteroaryl or heteroarylsubstituted with alkyl, acyl, alkoxy, halo, hydroxyl, nitro, cyano,dialkylamino, alkylsulfonyl, alkylsulfonamido, alkylthio, carboxyalkyl,carboxamido, aminocarbonyl, lower-alkylaminocarbonyl- (e.g.methylaminocarbonyl- or ethylaminocarbonyl-),di(lower-alkyl)aminocarbonyl- (e.g. dimethylaminocarbonyl- ordiethylaminocarbonyl-), aryl, or heteroaryl.

More preferably, R₅, R₆, R₇, and R₈ are independently chosen fromhydrogen, amino, alkylamino, hydroxyl, halogen (particularly chloro andfluoro), C₁-C₄ alkyl (particularly methyl), C₁-C₄ haloalkyl(particularly trifluoromethyl), C₁-C₄ alkoxy (particularly methoxy),C₁-C₄ haloalkoxy and cyano. More preferably, R₅, R₆, R₇, and R₈ aremethoxy, hydrogen, cyano, or halo (especially Cl, F). Further preferredfor each of the specific substituents: R₅ is amino, alkylamino,trifluoromethyl, hydrogen or halo; R₆ is hydrogen, alkyl (particularly,methyl) or halo; R₇ is hydrogen, halo, alkyl (particularly, methyl),alkoxy (particularly, methoxy), cyano, or trifluoromethyl; and R₈ ishydrogen or halo. Still further preferred are the compounds where onlyone of R₅, R₆, R₇, and R₈ is not hydrogen, especially R₇. More preferredare the compounds where R₅, R₆, and R₈ are hydrogen and R₇ is cyano,methoxy or halogen (especially Cl, F).

Preferred Salt Forms

Preferred compounds will generally be capable of forming acid additionsalts (i.e., will comprise a site which reacts with a pharmaceuticallyacceptable acid to form an acid addition salt.) The present inventionincludes pharmaceutically acceptable acid addition salts of thecompounds of Formula I. Acid addition salts of the present compounds areprepared in a standard manner in a suitable solvent from the parentcompound and an excess of an acid, such as hydrochloric, hydrobromic,sulfuric, phosphoric, acetic, maleic, succinic or methanesulfonic.Preferred salt forms are hydrochloric, phosphoric, and oxalic acid saltswith the hydrochloric acid salt form being especially preferred.

The salts and/or solvates of the compounds of the formula (I) which arenot pharmaceutically acceptable may be useful as intermediates in thepreparation of pharmaceutically acceptable salts and/or solvates ofcompounds of formula (I) or the compounds of the formula (I) themselves,and as such form another aspect of the present invention.

Preferred Subgenus

In a particularly preferred subgenus of compounds of Formula I, R₁ isbenzyl, halobenzyl, methoxybenzyl-, cyanobenzyl, or naphthalenylmethyl-;R₂ is ethyl or propyl; R_(2′) is hydrogen; R₅ is hydrogen; R₆ ishydrogen; R₇ is halo, cyano, methoxy or hydrogen; R₈ is hydrogen; andR₁₂ is —NR₄(COR₃) wherein R₃ is optionally substituted aryl (preferably,halophenyl, halomethylphenyl-, methylenedioxyphenyl-, methoxyphenyl-,ethoxyphenyl-, cyanophenyl- or phenyl substituted with lower-acyl orlower-alkylaminocarbonyl-, e.g. methylaminocarbonyl- orethylaminocarbonyl-, or di(lower-alkyl)aminocarbonyl-, e.g.dimethylaminocarbonyl- or diethylaminocarbonyl-; and R₄ is R₁₆-alkylene-wherein R₁₆ is hydroxyl, di(C₁-C₄)alkylamino-, (C₁-C₄ alkyl)amino-,amino, pyrrolidino, piperidino, imidazolyl and morpholino (morepreferably in such embodiments, R₁ is benzyl, halobenzyl, methoxybenzyl,cyanobenzyl, or naphthalenylmethyl; and R₂ is propyl (especially i- orc-propyl).

In another particularly preferred subgenus of compounds of Formula I, R₁is benzyl, halobenzyl, methoxybenzyl-, cyanobenzyl, ornaphthalenylmethyl-; R₂ is ethyl or propyl; R_(2′) is hydrogen; R₅ ishydrogen; R₆ is hydrogen; R₇ is halo, cyano, methoxy or hydrogen; R₈ ishydrogen; R₁₂ is —NR₄(CH₂R_(3b)) wherein R₄ is R₁₆-alkylene- wherein R₁₆is hydroxyl, di(C₁-C₄)alkylamino-, (C₁-C₄ alkyl)amino-, amino,pyrrolidino, piperidino, imidazolyl or morpholino.; and R_(3b) isoptionally substituted aryl.

In a particularly preferred subgenus of compounds of Formula I, R₁ isbenzyl, halobenzyl, methoxybenzyl, cyanobenzyl, or naphthalenylmethyl;R₂ is chosen from ethyl or propyl; R_(2′) is hydrogen; R₅ is hydrogen;R₆ is hydrogen; R₇ is halo, cyano, methoxy or hydrogen; R₈ is hydrogen;and R₁₂ is optionally substituted imidazolinyl of the above formulawherein R₁₀, R_(10′), R₁₄ and R_(14′) are independently hydrogen oroptionally substituted alkyl (preferably optionally substituted C₁-C₄alkyl); and R₉ is optionally substituted phenyl (preferably, halophenyl,halomethylphenyl, tolyl, or methylenedioxyphenyl). More preferably insuch embodiments R₁ is benzyl, methoxybenzyl, or cyanobenzyl; R₂ ispropyl (especially i- or c-propyl); and R₁₆ is amino.

In a particularly preferred subgenus of compounds of Formula I, R₁ isbenzyl, halobenzyl, methoxybenzyl, cyanobenzyl, or naphthalenylmethyl;R₂ is chosen from ethyl or propyl; R_(2′) is hydrogen; R₅ is hydrogen;R₆ is hydrogen; R₇ is halo, cyano, methoxy or hydrogen; R₈ is hydrogen;and R₁₂ is optionally substituted imidazole of the above formula whereinR₁₃ is hydrogen and R_(13′) is hydrogen or optionally substituted alkyl(preferably optionally substituted C₁-C₄ alkyl); and R₉ is optionallysubstituted aryl (preferably, halophenyl, halomethylphenyl, tolyl, ormethylenedioxyphenyl). More preferably, R₁₃ is hydrogen and R_(13′) isaminomethyl, aminoethyl aminopropyl, acetylamino-methyl,acetylaminoethyl, benzyloxycarbonylamino-methyl orBenzyloxycarbonylamino-ethyl. More preferably in such embodiments R₁ isbenzyl, methoxybenzyl, or cyanobenzyl; R₂ is propyl (especially i- orc-propyl); and R₁₆ is amino.

When R₁₂ is —N(R₄)(SO₂R_(3a)), R₁ is most preferably chosen from C₁-C₄alkyl, benzyl, substituted benzyl and substituted phenyl; R₂ is C₁-C₄alkyl; R_(2′) is hydrogen; R_(3a) is chosen from substituted phenyl andnaphthyl; R₄ is R₁₆-alkylene-; R₇ is hydrogen, fluoro, methyl or chloro;R₅, R₆ and R₈ are hydrogen; and R₁₆ is chosen from hydroxyl,di(C₁-C₄)amino, (C₁-C₄ alkyl)amino, amino, pyrrolidino, piperidino,imidazolyl and morpholino.

When R₁₂ is —NHR₄ or —N(R₄)(CH₂R_(3b)), R₁ is preferably chosen fromhydrogen, optionally substituted C₁-C₄ alkyl, optionally substitutedbenzyl, optionally substituted phenyl, and optionally substitutednaphthalenylmethyl; R₂ is optionally substituted C₁-C₄ alkyl and R_(2′)is hydrogen; R_(3b) is chosen from optionally substituted alkyl;optionally substituted phenyl; biphenylyl, optionally substitutedaralkyl; and optionally substituted heterocyclyl; and R₄ is chosen fromhydrogen, optionally substituted C₁-C₄ alkyl; cyclohexyl; optionallysubstituted phenyl; optionally substituted benzyl; heterocyclyl;heteroarylmethyl; heteroarylethyl; and heteroarylpropyl. Morepreferably, R₄ is R₁₆-alkylene-, wherein R₁₆ is hydroxyl,di(C₁-C₄)alkylamino-, (C₁-C₄ alkyl)amino-, amino, C₁-C₄ alkoxy-, orN-heterocyclyl.

When R₁₂ is —NHR₄ or —N(R₄)(CH₂R_(3b)), R₁ is most preferably chosenfrom C₁-C₄ alkyl, optionally substituted benzyl, and optionallysubstituted phenyl (more preferably optionally substituted benzyl, e.g.,benzyl, cyanobenzyl); R₂ is optionally substituted C₁-C₄ alkyl (morepreferably propyl, especially i- or c-propyl); R_(2′) is hydrogen;R_(3b) is chosen from optionally substituted phenyl, optionallysubstituted heterocyclyl and naphthyl; R₄ is chosen from hydrogen,optionally substituted benzyl, optionally substituted heterocyclyl andR₁₆-alkylene-; R₆ and R₇ are chosen from halo, cyano, methoxy orhydrogen; R₅ and R₈ are hydrogen; and R₁₆ is chosen from di(C₁-C₄alkylamino)-, (C₁-C₄ alkyl)amino-, amino, pyrrolidinyl, piperidinyl,imidazolyl and morpholinyl.

In a particularly preferred subgenus of compounds of Formula I, R₁ isbenzyl, halobenzyl (especially Cl-benzyl and F-benzyl), methoxybenzyl-,cyanobenzyl, or naphthalenylmethyl-; R₂ is ethyl or propyl; R_(2′) ishydrogen; R₅ is hydrogen; R₆ is hydrogen; R₇ is halo, cyano, methoxy orhydrogen; R₈ is hydrogen; and R₁₂ is —NHR₄ wherein R₄ is hydrogen (morepreferably in such embodiments, R₁ is benzyl, halobenzyl, cyanobenzyl;and R₂ is propyl, especially i-propyl or c-propyl).

When R_(3b) is present, it is most preferably chosen from phenylsubstituted with one or more halo, methyl, methoxy, cyano,trifluoromethyl, trifluoromethoxy, carboxy, and or methoxycarbonylgroups [e.g., halophenyl, polyhalophenyl, tolyl, dimethylphenyl,methoxyphenyl, dimethoxyphenyl, cyanophenyl, trifluoromethylphenyl,trifluoromethoxyphenyl, bis(trifluoromethyl)phenyl, carboxyphenyl,t-butylphenyl, methoxycarbonylphenyl]; piperidinyl and naphthyl.

Particularly preferred compounds include:

-   N-(3-Amino-propyl)-N-{1-[3-(3-cyano-benzyl)-7-hydroxy-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-3-fluoro-4-methyl-benzamide;-   4-Acetyl-N-(3-amino-propyl)-N-[1-(3-benzyl-7-methoxy-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-benzamide;-   2-{1-[4-(2-Amino-ethyl)-2-p-tolyl-imidazol-1-yl]-2-methyl-propyl}-3-benzyl-4-oxo-4H-chromene-7-carbonitrile;-   3-Benzyl-2-[1-(4,4-dimethyl-2-p-tolyl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-4-oxo-4H-chromene-7-carbonitrile;-   Benzo[1,3]dioxole-5-carboxylic acid    (3-amino-propyl)-{1-[7-chloro-3-(3-cyano-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-amide;-   N-(3-Amino-propyl)-N-{1-[7-chloro-3-(3-cyano-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methyl-benzamide;-   N-(3-Amino-propyl)-N-[1-(7-chloro-3-naphthalen-1-ylmethyl-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-2-methoxy-acetamide;-   4-Acetyl-N-(3-amino-propyl)-N-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-benzamide;-   N-(3-Amino-propyl)-N-{1-[7-chloro-3-(3-cyano-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-3-fluoro-4-methyl-benzamide;-   Benzo[1,3]dioxole-5-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   Benzo[1,3]dioxole-5-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   3-Benzyl-2-[1-(4,4-dimethyl-2-p-tolyl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-7-hydroxy-chromen-4-one;-   N-(3-Amino-propyl)-N-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-3-fluoro-4-methyl-benzamide;-   N-(3-Amino-propyl)-N-[1-(3-benzyl-7-methoxy-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-3-fluoro-4-methyl-benzamide;-   N-(3-Amino-propyl)-N-[1-(3-benzyl-7-methoxy-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamide;-   3-Benzyl-2-[1-(4,4-dimethyl-2-p-tolyl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-7-methoxy-chromen-4-one;-   3-Benzyl-7-fluoro-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,4-dimethyl-4,5-dihydro-imidazol-1-yl]-2-methyl-propyl}-chromen-4-one;-   3-Benzyl-2-[1-(4,4-dimethyl-2-p-tolyl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-7-fluoro-chromen-4-one;-   3-Benzyl-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,4-dimethyl-4,5-dihydro-imidazol-1-yl]-2-methyl-propyl}-7-cyano-chromen-4-one;-   N-(3-Amino-propyl)-N-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamide;-   4-Acetyl-N-(3-amino-propyl)-N-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-benzamide;-   N-(3-Amino-propyl)-3-fluoro-N-{1-[7-fluoro-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methyl-benzamide;-   4-Acetyl-N-(3-amino-propyl)-N-{1-[7-chloro-3-(3-cyano-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-benzamide;-   (2-{1-[1-(3-Benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-2-p-tolyl-1H-imidazol-4-yl}-ethyl)-carbamic    acid benzyl ester;-   2-[1-(2-Benzo[1,3]dioxol-5-yl-4,4-dimethyl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-3-benzyl-7-cyano-chromen-4-one;-   4-Acetyl-N-(3-amino-propyl)-N-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-benzamide;-   N-(3-Amino-propyl)-N-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-3-fluoro-4-methyl-benzamide;-   N-(3-Amino-propyl)-N-{1-[7-fluoro-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methoxy-benzamide;-   Benzo[1,3]dioxole-5-carboxylic acid    (3-amino-propyl)-{1-[7-fluoro-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-amide;-   2-{1-[4-(2-Amino-ethyl)-2-p-tolyl-imidazol-1-yl]-2-methyl-propyl}-3-benzyl-7-chloro-chromen-4-one;-   N-(3-Amino-propyl)-N-[1-(3-benzyl-7-hydroxy-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-3-fluoro-4-methyl-benzamide;-   Benzo[1,3]dioxole-5-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   N-(3-Amino-propyl)-N-[1-(3-benzyl-7-hydroxy-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamide;-   N-(3-Amino-propyl)-N-{1-[7-fluoro-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-2-methoxy-acetamide;-   N-(3-Amino-propyl)-N-{1-[7-fluoro-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methyl-benzamide;-   N-(3-Amino-propyl)-N-[(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-cyclopropyl-methyl]-4-methyl-benzamide;-   2,3-Dihydro-benzo[1,4]dioxine-6-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   N-(3-Amino-propyl)-3-fluoro-N-{1-[7-cyano-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methyl-benzamide;-   3-Benzyl-7-chloro-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-chromen-4-one;-   3-Benzyl-7-fluoro-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,5-dihydro-imidazol-1-yl]-2-methyl-propyl}-chromen-4-one;-   2-[1-(4-Aminomethyl-2-p-tolyl-imidazol-1-yl)-2-methyl-propyl]-3-benzyl-7-chloro-chromen-4-one;-   3-Benzyl-7-methoxy-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-chromen-4-one;-   2-[1-(2-Benzo[1,3]dioxol-5-yl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-3-benzyl-7-chloro-chromen-4-one;-   4-Acetyl-N-(3-amino-propyl)-N-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-benzamide;-   4-Acetyl-N-(3-amino-propyl)-N-{1-[7-cyano-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-benzamide;-   3-Benzyl-7-chloro-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,5-dihydro-imidazol-1-yl]-2-methyl-propyl}-chromen-4-one;-   3-Benzyl-7-fluoro-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-chromen-4-one;-   2-[1-(2-Benzo[1,3]dioxol-5-yl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-3-benzyl-7-fluoro-chromen-4-one;-   N-(3-Amino-propyl)-N-{1-[7-cyano-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methyl-benzamide;-   Benzo[1,3]dioxole-5-carboxylic acid    (3-amino-propyl)-{1-[7-cyano-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-amide;-   3-(2-{1-[2-(3-Fluoro-4-methyl-phenyl)-4,5-dihydro-imidazol-1-yl]-2-methyl-propyl}-7-methoxy-4-oxo-4H-chromen-3-ylmethyl)-benzonitrile;-   3-{7-Chloro-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-4-oxo-4H-chromen-3-ylmethyl}-benzonitrile;-   3-{7-Methoxy-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-4-oxo-4H-chromen-3-ylmethyl}-benzonitrile;-   3-{7-Fluoro-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-4-oxo-4H-chromen-3-ylmethyl}-benzonitrile;-   N-(3-Amino-propyl)-N-{1-[7-chloro-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-3-fluoro-4-methyl-benzamide;-   N-(3-Amino-propyl)-N-{1-[3-(3-cyano-benzyl)-7-methoxy-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methyl-benzamide;-   N-(3-Amino-propyl)-N-{1-[3-(3-cyano-benzyl)-7-methoxy-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-3-fluoro-4-methyl-benzamide;-   3-{2-[1-(2-Benzo[1,3]dioxol-5-yl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-7-fluoro-4-oxo-4H-chromen-3-ylmethyl}-benzonitrile;-   3-Benzyl-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,5-dihydro-imidazol-1-yl]-2-methyl-propyl}-4-oxo-4H-chromene-7-carbonitrile;-   2-[1-(2-Benzo[1,3]dioxol-5-yl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-3-benzyl-4-oxo-4H-chromene-7-carbonitrile;-   3-(7-Chloro-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,5-dihydro-imidazol-1-yl]-2-methyl-propyl}-4-oxo-4H-chromen-3-ylmethyl)-benzonitrile;-   3-{2-[1-(2-Benzo[1,3]dioxol-5-yl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-7-methoxy-4-oxo-4H-chromen-3-ylmethyl}-benzonitrile;-   3-(7-Fluoro-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,5-dihydro-imidazol-1-yl]-2-methyl-propyl}-4-oxo-4H-chromen-3-ylmethyl)-benzonitrile;-   Benzo[1,3]dioxole-5-carboxylic acid    (3-amino-propyl)-{1-[7-cyano-3-(3-cyano-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-amide;-   Benzo[1,3]dioxole-5-carboxylic acid    (3-amino-propyl)-{1-[3-(3-cyano-benzyl)-7-methoxy-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-amide;-   Benzo[1,3]dioxole-5-carboxylic acid    (3-amino-propyl)-{1-[3-(3-cyano-benzyl)-7-fluoro-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-amide;-   N-(3-Amino-propyl)-N-{1-[3-(3-cyano-benzyl)-7-fluoro-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methyl-benzamide;-   Benzo[1,3]dioxole-5-carboxylic acid    (3-amino-propyl)-{1-[7-chloro-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-amide;-   N-(3-Amino-propyl)-N-{1-[7-cyano-3-(3-cyano-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methyl-benzamide;-   3-Benzyl-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-4-oxo-4H-chromene-7-carbonitrile;-   2-[1-(2-Benzo[1,3]dioxol-5-yl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-3-benzyl-7-methoxy-chromen-4-one;-   N-(3-Amino-propyl)-N-{1-[7-chloro-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-2-methoxy-acetamide;-   N-(3-Amino-propyl)-N-{1-[7-cyano-3-(3-cyano-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-3-fluoro-4-methyl-benzamide;-   N-(3-Amino-propyl)-N-{1-[3-(3-cyano-benzyl)-7-fluoro-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-3-fluoro-4-methyl-benzamide;-   2-{1-[4-(2-Amino-ethyl)-2-p-tolyl-imidazol-1-yl]-2-methyl-propyl}-7-chloro-3-(3-methoxy-benzyl)-chromen-4-one;-   2-{1-[4-(2-Amino-ethyl)-2-(3-fluoro-4-methyl-phenyl)-imidazol-1-yl]-2-methyl-propyl}-3-benzyl-7-fluoro-chromen-4-one;-   2-{1-[4-(2-Amino-ethyl)-2-(3-fluoro-4-methyl-phenyl)-imidazol-1-yl]-2-methyl-propyl}-3-benzyl-4-oxo-4H-chromene-7-carbonitrile;-   2-{1-[4-(2-Amino-ethyl)-2-p-tolyl-imidazol-1-yl]-2-methyl-propyl}-3-benzyl-7-fluoro-chromen-4-one;-   2-{1-[4-(2-Amino-ethyl)-2-(3-fluoro-4-methyl-phenyl)-imidazol-1-yl]-2-methyl-propyl}-7-chloro-3-(3-methoxy-benzyl)-chromen-4-one;-   2-{1-[4-(2-Amino-ethyl)-2-(3-fluoro-4-methyl-phenyl)-imidazol-1-yl]-2-methyl-propyl}-3-benzyl-7-chloro-chromen-4-one;-   3-Benzyl-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,5-dihydro-imidazol-1-yl]-2-methyl-propyl}-7-methoxy-chromen-4-one;-   3-(2-{1-[4-(2-Amino-ethyl)-2-p-tolyl-imidazol-1-yl]-2-methyl-propyl}-7-chloro-4-oxo-4H-chromen-3-ylmethyl)-benzonitrile;-   3-(2-{1-[4-(2-Amino-ethyl)-2-(3-fluoro-4-methyl-phenyl)-imidazol-1-yl]-2-methyl-propyl}-7-chloro-4-oxo-4H-chromen-3-ylmethyl)-benzonitrile;-   2-{1-[4-(2-Amino-ethyl)-2-p-tolyl-imidazol-1-yl]-2-methyl-propyl}-7-fluoro-3-(3-methoxy-benzyl)-chromen-4-one;-   2-{1-[4-(2-Amino-ethyl)-2-(3-fluoro-4-methyl-phenyl)-imidazol-1-yl]-2-methyl-propyl}-3-(3-cyano-benzyl)-4-oxo-4H-chromene-7-carbonitrile;-   N-(3-Amino-propyl)-3-fluoro-N-{1-[7-hydroxy-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methyl-benzamide;-   2-{1-[4-(2-Amino-ethyl)-2-p-tolyl-imidazol-1-yl]-2-methyl-propyl}-3-(3-methoxy-benzyl)-4-oxo-4H-chromene-7-carbonitrile;-   N-(3-Amino-propyl)-N-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-ethoxy-benzamide;-   N-(3-Amino-propyl)-N-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-6-trifluoromethyl-nicotinamide;-   N-(3-Amino-propyl)-N-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-6-trifluoromethyl-nicotinamide;-   N-(3-Amino-propyl)-N-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-isonicotinamide;-   N-(3-Amino-propyl)-N-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-cyano-benzamide;-   4-Acetylamino-N-(3-amino-propyl)-N-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-benzamide;-   N-(3-Amino-propyl)-N-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-6-trifluoromethyl-nicotinamide;-   Benzo[1,2,3]thiadiazole-5-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   Benzo[1,2,3]thiadiazole-5-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   4-Acetylamino-N-(3-amino-propyl)-N-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-benzamide;-   Benzo[1,2,3]thiadiazole-5-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   N-(3-Amino-propyl)-N-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-nicotinamide;-   N-(3-Amino-propyl)-N-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methoxy-benzamide;-   Benzo[1,2,3]thiadiazole-5-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   5-Methyl-pyrazine-2-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   N-(3-Amino-propyl)-N-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-3-dimethylamino-benzamide;-   2-[1-(2-Benzo[1,3]dioxol-5-yl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-3-(3-methoxy-benzyl)-4-oxo-4H-chromene-7-carbonitrile;-   7-Chloro-3-(3-methoxy-benzyl)-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-chromen-4-one;-   7-Chloro-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,5-dihydro-imidazol-1-yl]-2-methyl-propyl}-3-(3-methoxy-benzyl)-chromen-4-one;-   2-[1-(2-Benzo[1,3]dioxol-5-yl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-7-chloro-3-(3-methoxy-benzyl)-chromen-4-one;-   7-Fluoro-3-(3-methoxy-benzyl)-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-chromen-4-one;-   7-Fluoro-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,5-dihydro-imidazol-1-yl]-2-methyl-propyl}-3-(3-methoxy-benzyl)-chromen-4-one;-   2-{1-[4-(2-Amino-ethyl)-2-(3-fluoro-4-methyl-phenyl)-imidazol-1-yl]-2-methyl-propyl}-7-fluoro-3-(3-methoxy-benzyl)-chromen-4-one;-   2-{1-[4-(2-Acetylamino-ethyl)-2-(3-fluoro-4-methyl-phenyl)-imidazol-1-yl]-2-methyl-propyl}-3-(3-methoxy-benzyl)-4-oxo-4H-chromene-7-carboxylic    acid amide;-   3-(2-{1-[4-(2-Amino-ethyl)-2-(3-fluoro-4-methyl-phenyl)-imidazol-1-yl]-2-methyl-propyl}-7-fluoro-4-oxo-4H-chromen-3-ylmethyl)-benzonitrile;-   N-{1-[1-(3-Benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-2-p-tolyl-1H-imidazol-4-ylmethyl}-acetamide;-   Benzo[b]thiophene-2-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   1-Methyl-1H-indole-2-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   5-tert-Butyl-2-methyl-2H-pyrazole-3-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   2,5-Dimethyl-2H-pyrazole-3-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   5-tert-Butyl-2-methyl-2H-pyrazole-3-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   2,5-Dimethyl-2H-pyrazole-3-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   2,5-Dimethyl-2H-pyrazole-3-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   5-Methyl-2H-pyrazole-3-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   5-Methyl-2H-pyrazole-3-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   Furan-2-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   Furan-2-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   2,5-Dimethyl-furan-3-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   2,5-Dimethyl-furan-3-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   5-Methyl-thiophene-2-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   5-Methyl-thiophene-2-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   5-Methyl-isoxazole-3-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   5-Methyl-2-trifluoromethyl-furan-3-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   5-Methyl-isoxazole-3-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   5-Methyl-isoxazole-3-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   Benzo[c]isoxazole-3-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   Benzo[c]isoxazole-3-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   1-Methyl-1H-pyrrole-2-carboxylic acid    (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   1-Methyl-1H-imidazole-4-carboxylic acid    (3-amino-propyl)-[(R)-1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   N-(3-Amino-propyl)-N-[(R)-1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-3-dimethylamino-benzamide;-   5-Methyl-2-trifluoromethyl-furan-3-carboxylic acid    (3-amino-propyl)-[(R)-1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   5-Methyl-isoxazole-3-carboxylic acid    (3-amino-propyl)-[(R)-1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   1-Methyl-1H-imidazole-4-carboxylic acid    (3-amino-propyl)-[(R)-1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   1-Methyl-1H-pyrrole-2-carboxylic acid    (3-amino-propyl)-[(R)-1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;-   Benzo[c]isoxazole-3-carboxylic acid    (3-amino-propyl)-[(R)-1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;    and-   5-Methyl-isoxazole-3-carboxylic acid    (3-amino-propyl)-[(R)-1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide.

UTILITY, TESTING AND ADMINISTRATION

General Utility

Once made, the compounds of the invention find use in a variety ofapplications involving alteration of mitosis. As will be appreciated bythose skilled in the art, mitosis may be altered in a variety of ways;that is, one can affect mitosis either by increasing or decreasing theactivity of a component in the mitotic pathway. Stated differently,mitosis may be affected (e.g., disrupted) by disturbing equilibrium,either by inhibiting or activating certain components. Similarapproaches may be used to alter meiosis.

In a preferred embodiment, the compounds of the invention are used toinhibit mitotic spindle formation, thus causing prolonged cell cyclearrest in mitosis. By “inhibit” in this context is meant decreasing orinterfering with mitotic spindle formation or causing mitotic spindledysfunction. By “mitotic spindle formation” herein is meant organizationof microtubules into bipolar structures by mitotic kinesins. By “mitoticspindle dysfunction” herein is meant mitotic arrest and monopolarspindle formation.

The compounds of the invention are useful to bind to, and/or inhibit theactivity of, a mitotic kinesin, KSP. In a preferred embodiment, the KSPis human KSP, although the compounds may be used to bind to or inhibitthe activity of KSP kinesins from other organisms. In this context,“inhibit” means either increasing or decreasing spindle pole separation,causing malformation, i.e., splaying, of mitotic spindle poles, orotherwise causing morphological perturbation of the mitotic spindle.Also included within the definition of KSP for these purposes arevariants and/or fragments of KSP. See U.S. Pat. No. 6,437,115, herebyincorporated by reference in its entirety. The compounds of theinvention have been shown to have specificity for KSP. However, thepresent invention includes the use of the compounds to bind to ormodulate other mitotic kinesins.

The compounds of the invention are used to treat cellular proliferationdiseases. Such disease states which can be treated by the compounds,compositions and methods provided herein include, but are not limitedto, cancer (further discussed below), autoimmune disease, fungaldisorders, arthritis, graft rejection, inflammatory bowel disease,cellular proliferation induced after medical procedures, including, butnot limited to, surgery, angioplasty, and the like. Treatment includesinhibiting cellular proliferation. It is appreciated that in some casesthe cells may not be in an abnormal state and still require treatment.Thus, in one embodiment, the invention herein includes application tocells or individuals afflicted or subject to impending affliction withany one of these disorders or states.

The compounds, compositions and methods provided herein are particularlydeemed useful for the treatment of cancer including solid tumors such asskin, breast, brain, cervical carcinomas, testicular carcinomas, etc.More particularly, cancers that may be treated by the compounds,compositions and methods of the invention include, but are not limitedto: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma,liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung:bronchogenic carcinoma (squamous cell, undifferentiated small cell,undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar)carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatoushamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cellcarcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach(carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma,insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), smallbowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor(nephroblastoma), lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma (pinealoma), glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma (serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma),fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia (acuteand chronic), acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignantlymphoma); Skin: malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma.Thus, the term “cancerous cell” as provided herein, includes a cellafflicted by any one of the above identified conditions.

Testing

For assay of KSP-modulating activity, generally either KSP or a compoundaccording to the invention is non-diffusably bound to an insolublesupport having isolated sample receiving areas (e.g., a microtiterplate, an array, etc.). The insoluble support may be made of anycomposition to which the sample can be bound, is readily separated fromsoluble material, and is otherwise compatible with the overall method ofscreening. The surface of such supports may be solid or porous and ofany convenient shape. Examples of suitable insoluble supports includemicrotiter plates, arrays, membranes and beads. These are typically madeof glass, plastic (e.g., polystyrene), polysaccharides, nylon ornitrocellulose, Teflon™, etc. Microtiter plates and arrays areespecially convenient because a large number of assays can be carriedout simultaneously, using small amounts of reagents and samples. Theparticular manner of binding of the sample is not crucial so long as itis compatible with the reagents and overall methods of the invention,maintains the activity of the sample and is nondiffusable. Preferredmethods of binding include the use of antibodies (which do notsterically block either the ligand binding site or activation sequencewhen the protein is bound to the support), direct binding to “sticky” orionic supports, chemical crosslinking, the synthesis of the protein oragent on the surface, etc. Following binding of the sample, excessunbound material is removed by washing. The sample receiving areas maythen be blocked through incubation with bovine serum albumin (BSA),casein or other innocuous protein or other moiety.

The compounds of the invention may be used on their own to inhibit theactivity of a mitotic kinesin, particularly KSP. In one embodiment, acompound of the invention is combined with KSP and the activity of KSPis assayed. Kinesin (including KSP) activity is known in the art andincludes one or more kinesin activities. Kinesin activities include theability to affect ATP hydrolysis; microtubule binding; gliding andpolymerization/depolymerization (effects on microtubule dynamics);binding to other proteins of the spindle; binding to proteins involvedin cell-cycle control; serving as a substrate to other enzymes, such askinases or proteases; and specific kinesin cellular activities such asspindle pole separation.

Methods of performing motility assays are well known to those of skillin the art. (See e.g., Hall, et al. (1996), Biophys. J., 71: 3467-3476,Turner et al., 1996, Anal. Biochem. 242 (1):20-5; Gittes et al., 1996,Biophys. J. 70(1): 418-29; Shirakawa et al., 1995, J. Exp. Biol. 198:1809-15; Winkelmann et al., 1995, Biophys. J. 68: 2444-53; Winkelmann etal., 1995, Biophys. J. 68: 72S.)

Methods known in the art for determining ATPase hydrolysis activity alsocan be used. Preferably, solution based assays are utilized. U.S. Pat.No. 6,410,254, hereby incorporated by reference in its entirety,describes such assays. Alternatively, conventional methods are used. Forexample, P₁ release from kinesin can be quantified. In one preferredembodiment, the ATPase hydrolysis activity assay utilizes 0.3 M PCA(perchloric acid) and malachite green reagent (8.27 mM sodium molybdateII, 0.33 mM malachite green oxalate, and 0.8 mM Triton X-100). Toperform the assay, 10 μL of the reaction mixture is quenched in 90 μL ofcold 0.3 M PCA. Phosphate standards are used so data can be converted tomM inorganic phosphate released. When all reactions and standards havebeen quenched in PCA, 100 μL of malachite green reagent is added to therelevant wells in e.g., a microtiter plate. The mixture is developed for10-15 minutes and the plate is read at an absorbance of 650 nm. Ifphosphate standards were used, absorbance readings can be converted tomM P₁ and plotted over time. Additionally, ATPase assays known in theart include the luciferase assay.

ATPase activity of kinesin motor domains also can be used to monitor theeffects of agents and are well known to those skilled in the art. In oneembodiment ATPase assays of kinesin are performed in the absence ofmicrotubules. In another embodiment, the ATPase assays are performed inthe presence of microtubules. Different types of agents can be detectedin the above assays. In a preferred embodiment, the effect of a agent isindependent of the concentration of microtubules and ATP. In anotherembodiment, the effect of the agents on kinesin ATPase can be decreasedby increasing the concentrations of ATP, microtubules or both. In yetanother embodiment, the effect of the agent is increased by increasingconcentrations of ATP, microtubules or both.

Compounds that inhibit the biochemical activity of KSP in vitro may thenbe screened in vivo. In vivo screening methods include assays of cellcycle distribution, cell viability, or the presence, morphology,activity, distribution, or number of mitotic spindles. Methods formonitoring cell cycle distribution of a cell population, for example, byflow cytometry, are well known to those skilled in the art, as aremethods for determining cell viability. See for example, U.S. Pat. No.6,437,115, hereby incorporated by reference in its entirety. Microscopicmethods for monitoring spindle formation and malformation are well knownto those of skill in the art (see, e.g., Whitehead and Rattner (1998),J. Cell Sci. 111:2551-61; Galgio et al, (1996) J. Cell Biol.,135:399-414), each incorporated herein by reference in its entirety.

The compounds of the invention inhibit the KSP kinesin. One measure ofinhibition is IC₅₀, defined as the concentration of the compound atwhich the activity of KSP is decreased by fifty percent relative to acontrol. Preferred compounds have IC₅₀'s of less than about 1 mM, withpreferred embodiments having IC₅₀'s of less than about 100 μM, with morepreferred embodiments having IC₅₀'s of less than about 10 μM, withparticularly preferred embodiments having IC₅₀'s of less than about 1μM, and especially preferred embodiments having IC₅₀'s of less thanabout 100 nM, and with the most preferred embodiments having IC₅₀'s ofless than about 10 nM. Measurement of IC₅₀ is done using an ATPase assaysuch as described herein.

Another measure of inhibition is K_(i). For compounds with IC₅₀'s lessthan 1 μM, the K_(i) or K_(d) is defined as the dissociation rateconstant for the interaction of the compounds described herein with KSP.Preferred compounds have K_(i)'s of less than about 100 μM, withpreferred embodiments having K_(i)'s of less than about 10 μM, andparticularly preferred embodiments having K_(i)'s of less than about 1μM and especially preferred embodiments having K_(i)'s of less thanabout 100 nM, and with the most preferred embodiments having K_(i)'s ofless than about 10 nM.

The K_(i) for a compound is determined from the IC₅₀ based on threeassumptions and the Michaelis-Menten equation. First, only one compoundmolecule binds to the enzyme and there is no cooperativity. Second, theconcentrations of active enzyme and the compound tested are known (i.e.,there are no significant amounts of impurities or inactive forms in thepreparations). Third, the enzymatic rate of the enzyme-inhibitor complexis zero. The rate (i.e., compound concentration) data are fitted to theequation:

$V = {V_{\max}{E_{0}\left\lbrack {I - \frac{\left( {E_{0} + I_{0} + {Kd}} \right) - \sqrt{\left( {E_{0} + I_{0} + {Kd}} \right)^{2} - {4E_{0}I_{0}}}}{2E_{0}}} \right\rbrack}}$where V is the observed rate, V_(max) is the rate of the free enzyme, I₀is the inhibitor concentration, E₀ is the enzyme concentration, andK_(d) is the dissociation constant of the enzyme-inhibitor complex.

Another measure of inhibition is GI₅₀, defined as the concentration ofthe compound that results in a decrease in the rate of cell growth byfifty percent. Preferred compounds have GI₅₀'s of less than about 1 mM;those having a GI₅₀ of less than about 20 are more preferred; thosehaving a GI₅₀ of less than about 10 μM more so; those having a GI₅₀ ofless than about 1 μM more so; those having a GI₅₀ of less than about 100nM more so; and those having a GI₅₀ of less than about 10 nM even moreso. Measurement of GI₅₀ is done using a cell proliferation assay such asdescribed herein. Compounds of this class were found to inhibit cellproliferation.

In vitro potency of small molecule inhibitors is determined, forexample, by assaying human ovarian cancer cells (SKOV3) for viabilityfollowing a 72-hour exposure to a 9-point dilution series of compound.Cell viability is determined by measuring the absorbance of formazon, aproduct formed by the bioreduction of MTS/PMS, a commercially availablereagent. Each point on the dose-response curve is calculated as apercent of untreated control cells at 72 hours minus backgroundabsorption (complete cell kill).

Anti-proliferative compounds that have been successfully applied in theclinic to treatment of cancer (cancer chemotherapeutics) have GI₅₀'sthat vary greatly. For example, in A549 cells, paclitaxel GI₅₀ is 4 nM,doxorubicin is 63 nM, 5-fluorouracil is 1 μM, and hydroxyurea is 500 μM(data provided by National Cancer Institute, Developmental TherapeuticProgram, http://dtp.nci.nih.gov/). Therefore, compounds that inhibitcellular proliferation, irrespective of the concentration demonstratinginhibition, may be useful.

To employ the compounds of the invention in a method of screening forcompounds that bind to KSP kinesin, the KSP is bound to a support, and acompound of the invention is added to the assay. Alternatively, thecompound of the invention is bound to the support and KSP is added.Classes of compounds among which novel binding agents may be soughtinclude specific antibodies, non-natural binding agents identified inscreens of chemical libraries, peptide analogs, etc. Of particularinterest are screening assays for candidate agents that have a lowtoxicity for human cells. A wide variety of assays may be used for thispurpose, including labeled in vitro protein-protein binding assays,electrophoretic mobility shift assays, immunoassays for protein binding,functional assays (phosphorylation assays, etc.) and the like.

The determination of the binding of the compound of the invention to KSPmay be done in a number of ways. In a preferred embodiment, the compoundis labeled, for example, with a fluorescent or radioactive moiety, andbinding is determined directly. For example, this may be done byattaching all or a portion of KSP to a solid support, adding a labeledtest compound (for example a compound of the invention in which at leastone atom has been replaced by a detectable isotope), washing off excessreagent, and determining whether the amount of the label is that presenton the solid support.

By “labeled” herein is meant that the compound is either directly orindirectly labeled with a label which provides a detectable signal,e.g., radioisotope, fluorescent tag, enzyme, antibodies, particles suchas magnetic particles, chemiluminescent tag, or specific bindingmolecules, etc. Specific binding molecules include pairs, such as biotinand streptavidin, digoxin and antidigoxin etc. For the specific bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures, as outlined above. The label can directly or indirectlyprovide a detectable signal.

In some embodiments, only one of the components is labeled. For example,the kinesin proteins may be labeled at tyrosine positions using ¹²⁵I, orwith fluorophores. Alternatively, more than one component may be labeledwith different labels; using ¹²⁵I for the proteins, for example, and afluorophor for the antimitotic agents.

The compounds of the invention may also be used as competitors to screenfor additional drug candidates. “Candidate agent” or “drug candidate” orgrammatical equivalents as used herein describe any molecule, e.g.,protein, oligopeptide, small organic molecule, polysaccharide,polynucleotide, etc., to be tested for bioactivity. They may be capableof directly or indirectly altering the cellular proliferation phenotypeor the expression of a cellular proliferation sequence, including bothnucleic acid sequences and protein sequences. In other cases, alterationof cellular proliferation protein binding and/or activity is screened.Screens of this sort may be performed either in the presence or absenceof microtubules. In the case where protein binding or activity isscreened, preferred embodiments exclude molecules already known to bindto that particular protein, for example, polymer structures such asmicrotubules, and energy sources such as ATP. Preferred embodiments ofassays herein include candidate agents which do not bind the cellularproliferation protein in its endogenous native state termed herein as“exogenous” agents. In another preferred embodiment, exogenous agentsfurther exclude antibodies to KSP.

Candidate agents can encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 100 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding andlipophilic binding, and typically include at least an amine, carbonyl,hydroxyl, ether, or carboxyl group, preferably at least two of thefunctional chemical groups. The candidate agents often comprise cyclicalcarbon or heterocyclic structures and/or aromatic or polyaromaticstructures substituted with one or more of the above functional groups.Candidate agents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, and/or amidification to producestructural analogs.

Competitive screening assays may be done by combining KSP and a drugcandidate in a first sample. A second sample comprises a compound of thepresent invention, KSP and a drug candidate. This may be performed ineither the presence or absence of microtubules. The binding of the drugcandidate is determined for both samples, and a change, or difference inbinding between the two samples indicates the presence of a drugcandidate capable of binding to KSP and potentially inhibiting itsactivity. That is, if the binding of the drug candidate is different inthe second sample relative to the first sample, the drug candidate iscapable of binding to KSP.

In a preferred embodiment, the binding of the candidate agent to KSP isdetermined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to KSP,such as an antibody, peptide, binding partner, ligand, etc. Undercertain circumstances, there may be competitive binding as between thecandidate agent and the binding moiety, with the binding moietydisplacing the candidate agent.

In one embodiment, the candidate agent is labeled. Either the candidateagent, or the competitor, or both, is added first to KSP for a timesufficient to allow binding, if present. Incubations may be performed atany temperature which facilitates optimal activity, typically between 4and 40° C.

Incubation periods are selected for optimum activity, but may also beoptimized to facilitate rapid high throughput screening. Typicallybetween 0.1 and 1 hour will be sufficient. Excess reagent is generallyremoved or washed away. The second component is then added, and thepresence or absence of the labeled component is followed, to indicatebinding.

In a preferred embodiment, the competitor is added first, followed bythe candidate agent. Displacement of the competitor is an indication thecandidate agent is binding to KSP and thus is capable of binding to, andpotentially inhibiting, the activity of KSP. In this embodiment, eithercomponent can be labeled. Thus, for example, if the competitor islabeled, the presence of label in the wash solution indicatesdisplacement by the agent. Alternatively, if the candidate agent islabeled, the presence of the label on the support indicatesdisplacement.

In an alternative embodiment, the candidate agent is added first, withincubation and washing, followed by the competitor. The absence ofbinding by the competitor may indicate the candidate agent is bound toKSP with a higher affinity. Thus, if the candidate agent is labeled, thepresence of the label on the support, coupled with a lack of competitorbinding, may indicate the candidate agent is capable of binding to KSP.

Inhibition is tested by screening for candidate agents capable ofinhibiting the activity of KSP comprising the steps of combining acandidate agent with KSP, as above, and determining an alteration in thebiological activity of KSP. Thus, in this embodiment, the candidateagent should both bind to KSP (although this may not be necessary), andalter its biological or biochemical activity as defined herein. Themethods include both in vitro screening methods and in vivo screening ofcells for alterations in cell cycle distribution, cell viability, or forthe presence, morpohology, activity, distribution, or amount of mitoticspindles, as are generally outlined above.

Alternatively, differential screening may be used to identify drugcandidates that bind to the native KSP, but cannot bind to modified KSP.

Positive controls and negative controls may be used in the assays.Preferably all control and test samples are performed in at leasttriplicate to obtain statistically significant results. Incubation ofall samples is for a time sufficient for the binding of the agent to theprotein. Following incubation, all samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples may be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g., albumin,detergents, etc which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

Administration

Accordingly, the compounds of the invention are administered to cells.By “cells” herein is meant any cell in which mitosis or meiosis can bealtered. By “administered” herein is meant administration of atherapeutically effective dose of a compound of the invention to a celleither in cell culture or in a patient. By “therapeutically effectivedose” herein is meant a dose that produces the effects for which it isadministered. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques. As is known in the art, adjustments for systemicversus localized delivery, route of administration, age, body weight,general health, sex, diet, time of administration, nature of theformulation, drug interaction, and the precise condition requiringtreatment and its severity may be necessary, and will be ascertainablewith routine experimentation by those skilled in the art. However, aneffective amount of a compound of Formula I for the treatment ofneoplastic growth (typically by intravenous administration), for examplecolon or breast carcinoma, will generally be in the range of 0.1 to 100(including 1 to 100) mg/m² of surface area of the recipient per dose ona once a week to once a month schedule and usually in the range of 2 to30 mg/m² of surface area of the recipient per dose on a once a week toonce a month schedule. An effective amount of a salt, solvate, orsolvate of a salt of a compound of Formula I may be determined as aproportion of the effective amount of the compound of Formula I per se.It is envisaged that similar dosages would be appropriate for treatmentof the other conditions referred to herein.

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals, and other organisms.Thus the methods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal, andin the most preferred embodiment the patient is human.

Compounds of the invention having the desired pharmacological activitymay be administered, preferably as a pharmaceutically acceptablecomposition comprising an pharmaceutical excipient, to a patient, asdescribed herein. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways as discussed below. Theconcentration of therapeutically active compound in the formulation mayvary from about 0.1-100 wt. %.

The agents may be administered alone or in combination with othertreatments, i.e., radiation, or other chemotherapeutic agents such asthe taxane class of agents that appear to act on microtubule formationor the camptothecin class of topoisomerase I inhibitors. When used,other chemotherapeutic agents may be administered before, concurrently,or after administration of a compound of the present invention. In oneaspect of the invention, a compound of the present invention isco-administered with one or more other chemotherapeutic agents. By“co-administer” it is meant that the present compounds are administeredto a patient such that the present compounds as well as theco-administered compound may be found in the patient's bloodstream atthe same time, regardless when the compounds are actually administered,including simultaneously.

The administration of the compounds and compositions of the presentinvention can be done in a variety of ways, including, but not limitedto, orally, subcutaneously, intravenously, intranasally, transdermally,intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally,or intraocularly. In some instances, for example, in the treatment ofwounds and inflammation, the compound or composition may be directlyapplied as a solution or spray.

Pharmaceutical dosage forms include a compound of formula I or apharmaceutically acceptable salt or solvate thereof, and one or morepharmaceutical excipients. As is known in the art, pharmaceuticalexcipients are secondary ingredients which function to enable or enhancethe delivery of a drug or medicine in a variety of dosage forms (e.g.:oral forms such as tablets, capsules, and liquids; topical forms such asdermal, opthalmic, and otic forms; suppositories; injectables;respiratory forms and the like). Pharmaceutical excipients include inertor inactive ingredients, synergists or chemicals that substantivelycontribute to the medicinal effects of the active ingredient. Forexample, pharmaceutical excipients may function to improve flowcharacteristics, product uniformity, stability, taste, or appearance, toease handling and administration of dose, for convenience of use, or tocontrol bioavailability. While pharmaceutical excipients are commonlydescribed as being inert or inactive, it is appreciated in the art thatthere is a relationship between the properties of the pharmaceuticalexcipients and the dosage forms containing them.

Pharmaceutical excipients suitable for use as carriers or diluents arewell known in the art, and may be used in a variety of formulations.See, e.g., Remington's Pharmaceutical Sciences, 18th Edition, A. R.Gennaro, Editor, Mack Publishing Company (1990); Remington: The Scienceand Practice of Pharmacy, 20th Edition, A. R. Gennaro, Editor,Lippincott Williams & Wilkins (2000); Handbook of PharmaceuticalExcipients, 3rd Edition, A. H. Kibbe, Editor, American PharmaceuticalAssociation, and Pharmaceutical Press (2000); and Handbook ofPharmaceutical Additives, compiled by Michael and Irene Ash, Gower(1995), each of which is incorporated herein by reference for allpurposes.

Oral solid dosage forms such as tablets will typically comprise one ormore pharmaceutical excipients, which may for example help impartsatisfactory processing and compression characteristics, or provideadditional desirable physical characteristics to the tablet. Suchpharmaceutical excipients may be selected from diluents, binders,glidants, lubricants, disintegrants, colors, flavors, sweetening agents,polymers, waxes or other solubility-retarding materials.

Compositions for intravenous administration will generally compriseintravenous fluids, i.e., sterile solutions of simple chemicals such assugars, amino acids or electrolytes, which can be easily carried by thecirculatory system and assimilated. Such fluids are prepared with waterfor injection USP.

Fluids used commonly for intravenous (IV) use are disclosed inRemington, the Science and Practice of Pharmacy [full citationpreviously provided], and include:

alcohol (e.g., in dextrose and water (“D/W”) [e.g., 5% dextrose] ordextrose and water [e.g., 5% dextrose] in normal saline solution(“NSS”); e.g. 5% alcohol);

synthetic amino acid such as Aminosyn, FreAmine, Travasol, e.g., 3.5 or7; 8.5; 3.5, 5.5 or 8.5% respectively;

ammonium chloride e.g., 2.14%;

dextran 40, in NSS e.g., 10% or in D5/W e.g., 10%;

dextran 70, in NSS e.g., 6% or in D5/W e.g., 6%;

dextrose (glucose, D5/W) e.g., 2.5-50%;

dextrose and sodium chloride e.g., 5-20% dextrose and 0.22-0.9% NaCl;

lactated Ringer's (Hartmann's) e.g., NaCl 0.6%, KCl 0.03%, CaCl₂ 0.02%;

lactate 0.3%;

mannitol e.g., 5%, optionally in combination with dextrose e.g., 10% orNaCl e.g., 15 or 20%;

multiple electrolyte solutions with varying combinations ofelectrolytes, dextrose, fructose, invert sugar Ringer's e.g., NaCl0.86%, KCl 0.03%, CaCl₂ 0.033%;

sodium bicarbonate e.g., 5%;

sodium chloride e.g., 0.45, 0.9, 3, or 5%;

sodium lactate e.g., ⅙ M; and

sterile water for injection

The pH of such fluids may vary, and will typically be from 3.5 to 8 suchas known in the art.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

EXAMPLES

All anhydrous solvents were purchased from Aldrich Chemical Company inSureSeal® containers. Reagents were added and aqueous extractionsperformed with single or multichannel pipettors. Filtrations wereperformed using Whatman/Polyfiltronics 24 well, 10 mL filtration blocks.Evaporation of volatile materials from the array was performed with aLabconco Vortex-Evaporator or by sweeping with a 4×6 nitrogen manifold.

Example 1

Synthesis of Compounds

a) 3-Phenyl-propionic acid 3-chloro-phenyl ester

To a solution of 3-chlorophenol (1, 50.1 g, 0.3 mol), triethylamine(TEA, 85 mL), and CH₂Cl₂ (500 mL) at 23° C. was added hydrocinnamoylchloride (2, 31 mL, 0.3 mol) over 5 minutes. After 30 minutes, thereaction mixture was concentrated in vacuo. The crude slurry was thendissolved in 10:1 hexanes:EtOAc (300 mL) and washed with 1 N NaOH (100mL) and brine (100 mL). The organic layer was dried (MgSO₄) and filteredthrough a plug of silica gel (10:1 hexanes:EtOAc rinse). The eluent wasconcentrated to afford 78 g of a slightly yellow oil, which was usedwithout any further purification.

b) 1-(4-Chloro-2-hydroxy-phenyl)-3-phenyl-propan-1-one

AlCl₃ (52 g, 0.39 mol) was added slowly over 15 minutes to ester 3 (78g, 0.3 mol) at 140° C. After an additional 15 minutes gas evolutionceased and the reaction mixture was poured into a 1 L beaker and allowedto cool to RT. The resulting solid was dissolved in CH₂Cl₂ (100 mL) andslowly quenched with 1 N HCl (200 mL). This mixture was diluted withEtOAc (600 mL) and the layers were separated. The organic layer waswashed with brine (100 mL), dried (MgSO₄), filtered, and concentrated.The crude oil was dissolved in 20:1 hexanes:EtOAc (500 mL) and flushedthrough a plug of silica gel (100% hexanes; 20:1 hexanes:EtOAc rinse).The filtrate was concentrated to provide a slightly brown oil, which waspurified by flash column chromatography (50:1 hexanes:EtOAc; 40:1hexanes:EtOAc; 30:1 hexanes:EtOAc; 20:1 hexanes:EtOAc) to yield 31.2 g(40%) of 4 as a white solid.

c) 2-tert-Butoxycarbonylamino-3-methyl-butyric acid5-chloro-2-(3-phenyl-propionyl)-phenyl ester

A solution of phenol 4 (23.18 g, 89.1 mmol), BOC-D-Valine (5, 21.29 g,98.05 mmol), HBTU (40.57 g, 107 mmol), TEA (37 mL, 265 mmol), and CH₂Cl₂(155 mL) were maintained at 23° C. for 5 hours. The reaction mixture wasdiluted with EtOAc (500 mL) and washed with saturated aqueous NH₄Cl(2×100 mL) and brine (100 mL). The organic layer was dried (MgSO₄),filtered, and concentrated. The resulting residue was purified by flashcolumn chromatography (100% hexanes; 50:1 hexanes:EtOAc; 40:1hexanes:EtOAc; 30:1 hexanes:EtOAc; 20:1 hexanes:EtOAc; 10:1hexanes:EtOAc) to yield 35.7 g (87%) of 6 as a yellow oil.

d)[1-(3-Benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-carbamicacid tert-butyl ester

A mixture of ester 6 (35.55 g, 79.17 mmol), K₂CO₃ (21.8 g, 158.4 mmol),and DMF (264 mL) was placed into a 140° C. oil bath. After 30 mins, thereaction mixture was quenched with H₂O (300 mL) and extracted with Et₂O(3×200 mL). The organic layer was washed with brine (100 mL), dried(MgSO₄), filtered, and concentrated. The resulting residue was purifiedby flash column chromatography (10:1 hexanes:EtOAc) to yield 3.5 g(10.2%) of 7.

e) 2-(1-Amino-2-methyl-propyl)-3-benzyl-7-chloro-chromen-4-one

Chromenone 7 (1.8 g, 4.18 mmol) and TFA:H₂O (97.5:2.5, 30 mL) wasmaintained at 23° C. for 1 h. The reaction mixture was concentrated. Theresidue was dissolved in EtOAc (100 mL) and washed with 1 N NaOH (25 mL)and brine (25 mL). The organic layer was dried (MgSO₄), filtered, andconcentrated to provide a colorless oil, which was used without furtherpurification.

f)N-{2-[1-(3-Benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propylamino]-1,1-dimethyl-ethyl}-4-methyl-benzamide

Chromenone 8 (72 mg, 0.22 mmol), aldehyde 9 (65 mg, 0.32 mmol),Na(OAc)₃BH (184 mg, 0.87 mmol), and CH₂Cl₂ (1 mL) was maintained at 23°C. for 3 h. The reaction mixture was diluted with EtOAc (20 mL) andwashed with 1 N NaOH (5 mL) and brine (5 mL). The organic layer wasdried (MgSO₄), filtered, and concentrated. The resulting residue waspurified by flash column chromatography (3:1 hexanes:EtOAc) to yield 80mg (70%) of 10 as a white solid.

g)3-Benzyl-7-chloro-2-[1-(4,4-dimethyl-2-p-tolyl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-chromen-4-one

Chromenone 10 (80 mg, 0.15 mmol), POCl₃ (0.1 mL, 1.1 mmol), and PhMe (1mL) were heated to 110° C. After 3.5 h, an additional portion of POCl₃(0.1 mL, 1.1 mmol) was added. After 1 h, the reaction mixture wasdiluted with EtOAc (20 mL) and washed with 1 N NaOH (10 mL) and brine(10 mL). The organic layer was dried (MgSO₄), filtered, andconcentrated. The resulting residue was purified by flash columnchromatography (20:1 CHCl₃:MeOH) to yield 50 mg (65%) of 11 as a whitesolid.

Example 2

Synthesis of Compounds

a) Preparation of{3-[1-(3-Benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propylamino]-propyl}-carbamicacid tert-butyl ester

Chromenone 8 (420 mg, 1.24 mmol), aldehyde 12 (280 mg, 1.6 mmol),NaCN(OAc)₃BH (790 mg, 3.7 mmol), and CH₂Cl₂ (4.1 mL) was maintained at23° C. for 3 h. The reaction mixture was diluted with EtOAc (20 mL) andwashed with 1 N NaOH (5 mL) and brine (5 mL). The organic layer wasdried (MgSO₄), filtered, and concentrated. The resulting residue waspurified by flash column chromatography (5:1 hexanes:EtOAc; 3:1hexanes:EtOAc) to yield 460 mg (75%) of 13 as a viscous oil.

b) Preparation of{3-[[1-(3-Benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-(4-methyl-benzoyl)-amino]-propyl}-carbamicacid tert-butyl ester

To a solution of chromenone 13 (1.3 g, 2.6 mmol), diisoproylethylamine(DIEA, 1.8 mL), and CH₂Cl₂ (7.5 mL) at 23° C. was added p-toluoylchloride (0.7 mL, 5.22 mmol). After 2.5 h, the reaction mixture wasdiluted with EtOAc (100 mL) and washed with saturated aqueous NaHCO₃(2×20 mL) and brine (20 mL). The organic layer was dried (MgSO₄),filtered, and concentrated. The resulting residue was purified by flashcolumn chromatography (3:1 hexanes:EtOAc) to yield 1.43 g (89%) of 14 asa colorless oil.

c) Preparation ofN-(3-Amino-propyl)-N-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamide

Chromenone 14 (1.43 g, 2.32 mmol) and TFA:H₂O (97.5:2.5, 30 mL) wasmaintained at 23° C. for 1 h. The reaction mixture was concentrated. Theresidue was dissolved in EtOAc (100 mL) and washed with 1 N NaOH (25 mL)and brine (25 mL). The organic layer was dried (MgSO₄), filtered, andconcentrated to provide a white solid which was deemed >95% pure by ¹HNMR and LCMS analysis.

Using procedures analogous to those set forth above in Example 2, thefollowing compound was prepared.

Structure LRMS (MH) m/z

471.2

Example 3

Synthesis of Compounds

Carbonyldiimidazole (9.14 g, 56.37 mmol) was added slowly to a r.t.solution of BOC-D-Valine (5, 12.25 g, 56.37 mmol) and THF (185 mL).After 1 h, the solution was washed with 50% aqueous NaCl (200 mL),followed by brine (2×200 mL). The organic layer was dried (MgSO₄),filtered, and concentrated to provide a white solid, which was usedwithout further purification.

(2-Bromoethyl)benzene (2.51 mL, 18.38 mmol), magnesium turnings (477 mg,19.62 mmol), and THF (20 mL) were heated to 60° C. for 1 hour, thenallowed to cool to r.t. A solution of 16 (2.0 g, 9.19 mmol) and THF (20mL) was cooled to 0-5° C. The solution of the phenethyl magnesiumchloride was then added via syringe to the 0-5° C. solution of thevaline imidazole. The temperature was monitored by internal thermometerand was not allowed to exceed 15° C. The reaction mixture was maintainedat 0-5° C. for 1 hour. The reaction mixture was quenched with saturatedaqueous NH₄Cl (20 mL), and diluted with EtOAc (100 mL). The layers wereseparated and the organic layer was washed with brine (30 mL). Theorganic layer was dried (MgSO₄), filtered, and concentrated. Theresulting residue was purified by flash column chromatography (10:1hexanes:EtOAc; 5:1 hexanes:EtOAc) to yield 1.15 g (41%) of 17. LRMS(MH-tBuOCO) m/z 206.1.

LHMDS (1.M in THF, 9.53 mL, 3.3 equiv) was added slowly via syringe to a−78° C. solution of ketone 17 (882 mg, 2.89 mmol). After the additionwas complete the resulting light orange solution was maintained at −78°C. for 40 mins. Neat 4-chloro-2-fluorobenzoyl chloride (18, 460 μL, 2.89mmol) (assumed density to be 1.20 g/mL) was added dropwise via syringe.The reaction solution turned to an orange color and was maintained for40 mins. The reaction solution was quenched with saturated aqueous NH₄Cl(20 mL), and diluted with EtOAc (50 mL). The layers were separated andthe organic layer was washed with brine (30 mL). The organic layer wasdried (MgSO₄), filtered, and concentrated. The resulting residue waspurified by flash column chromatography (10:1 hexanes:EtOAc) to yield1.15 g (86%) of 19. LRMS (MH-HF) m/z 442.1.

A mixture of 19 (1.15 g, 2.49 mmol), K₂CO₃ (420 mg, 3.04 mmol), and DMF(12 mL) was maintained at r.t. for 30 mins. The yellow reaction solutionwas quenched with brine (50 mL), and diluted with Et₂O (50 mL). Thelayers were separated and the organic layer was washed with brine (2×50mL). The organic layer was dried (MgSO₄), filtered, and concentrated.The resulting residue was purified by flash column chromatography (10:1hexanes:EtOAc) to yield 20. LRMS (MH) m/z 442.1.

Using methods analogous to those described above in Examples 2 and 3,the compounds shown below were prepared.

Stereochemistry R₇ R R′ (R/S) Cl H H 3/1 Cl —CH₂CMe₂N═C(p-tol)— 3/1 ClH₂N(CH₂)₃— p-toluoyl 3/1 Cl H₂N(CH₂)₃— MeOCH₂C(O)— 3/1 Cl H₂N(CH₂)₃—p-Br-phenyl- 3/1 Cl H₂N(CH₂)₃— p-toluoyl R Cl H₂N(CH₂)₃— p-toluoyl S Cl—CH₂CMe₂N═C(p-tol)— R Cl —CH₂CMe₂N═C(p-tol)— S F H H 3/1 F H₂N(CH₂)₃—p-toluoyl 3/1 F H₂N(CH₂)₃— MeOCH₂C(O)— 3/1 F —CH₂CMe₂N═C(p-tol)— 3/1

Example 4

Alternative Methods for Preparation of Compounds

Ethylchloroformate (11.0 mL, 115 mmol) was added over 1 minute to a 0-5°C. solution of BOC-D-Valine (5, 25.0 g, 115 mmol), triethylamine (16.0mL, 115 mmol), and THF (145 mL) under N₂. The internal temperature ofthe reaction solution rose to 9° C. After 15 mins, a mixture ofdimethylhydroxylamine hydrochloride (13.46 g, 138 mmol), triethylamine(32.0 mL, 230 mmol), and THF (110 mL) was added over 5 minutes. Theinternal temperature rose to 17° C. Upon completion of addition, theice/H₂O bath was removed and the reaction solution maintained at 23° C.for 1 hour. The reaction solution was then concentrated. The cruderesidue was dissolved in EtOAc (200 mL) and washed with 1 N HCl (200 mL)and brine (100 mL). The organic layer was dried (MgSO₄), filtered, andconcentrated to provide 30 g (˜100%) of 21 as a colorless oil, which wasused without further purification.

(2-Bromoethyl)benzene (38.0 mL, 273 mmol), magnesium turnings (7.0 g,289 mmol), and Et₂O (500 mL) were mixed in a 1 L round-bottom flaskequipped with a reflux condenser at 23° C. under a N₂ atmosphere. After˜10 mins the reaction mixture begins to exotherm and the reactionmixture was allowed to progress to reflux with intermittent cooling withan ice/H₂O bath. After 1.5 hour, the Grignard reaction was complete andthe solution had cooled to 23° C. A solution of 21 (18.0 g, 82.7 mmol)and Et₂O (200 mL) was added via cannula to the 20° C. solution of thephenethylmagnesium bromide. The temperature was monitored by internalthermometer and was not allowed to exceed ˜30° C. The reaction mixturetemperature was monitored by an internal thermometer and regulated(20-30° C.) with an ice/H₂O bath. After 1 h at 23° C., the reactionmixture was quenched by pouring into 1 N HCl (300 mL). The layers wereseparated and the organic layer was washed with brine (100 mL). Theorganic layer was dried (MgSO₄), filtered, and concentrated. Theresulting residue was purified by flash column chromatography (10:1hexanes:EtOAc) to yield 13.4 g (53%) of 17. LRMS (MH-tBuOCO) m/z 206.1.

Lithium bis(trimethylsilyl)amide (LHMDS, 1.0 M in THF, 94.0 mL, 3.3equiv) was added slowly over ˜3 minutes via syringe to a −78° C.solution of ketone 17 (8.74 g, 28.62 mmol) and THF (100 mL). Thereaction solution temperature was monitored by an internal thermometer,and addition of the base was done at a rate sufficient to prevent thetemperature from exceeding −54° C. After the addition was complete theresulting solution was maintained at −78° C. for 30 mins. Neat4-chloro-2-fluorobenzoyl chloride (18, 4.58 mL, 28.62 mmol) (assumeddensity to be 1.20 g/mL) was added drop-wise over ˜1 minute via syringe(temperature rose from −78° C. to −59° C.). The reaction solution wasmaintained at −78° C. for 30 mins. The reaction solution was quenchedwith 1 N HCl (100 mL). The layers were separated and the organic layerwas washed with brine (100 mL). The organic layer was dried (MgSO₄),filtered, and concentrated. The resulting residue was used withoutfurther purification.

A mixture of the above crude product, K₂CO₃ (4.75 g, 34.34 mmol) and DMF(100 mL) was maintained at 23° C. for 30 mins. The reaction mixture wasquenched by addition of Et₂O (200 mL) and brine (200 mL). The layerswere separated and the organic layer was washed with brine (2×200 mL).The organic layer was dried (MgSO₄), filtered, and concentrated. Theresulting residue absorbed onto silica gel (CH₂Cl₂) and was purified byflash column chromatography (10:1 hexanes:EtOAc) to yield 11.5 g (91%for 2 steps) of 20 as a white solid. LRMS (MH) m/z 442.1.

Example 5

Methylmagnesium chloride (82.5 mL, 3.0 M in THF, 247 mmol) was addedover 5 mins to a 0-5° C. solution of 21 (17.95 g, 69.0 mmol) and THF(200 mL) under an N₂ atmosphere. The reaction mixture temperature wasmonitored by an internal thermometer, and addition of the Grignard wasdone at a rate sufficient to prevent the temperature from exceeding 19°C. Upon complete addition the cooling bath was removed and the reactionmixture was maintained at 23° C. for 2 hours. The reaction mixture wasthen quenched with 1 N HCl (100 mL). The layers were separated and theorganic layer was washed with brine (100 mL). The organic layer wasdried (MgSO₄), filtered, and concentrated to provide 12.5 g (84%) of 22as a white solid (>95% pure by ¹H NMR), which was used without furtherpurification.

LHMDS (128 mL, 1.0 M in THF) was added via syringe over 3 minutes to a−78° C. solution of ketone 22 (12.5 g, 58.1 mmol) and THF (200 mL). Thereaction solution temperature was monitored by an internal thermometer,and addition of the base was done at a rate sufficient to prevent thetemperature from exceeding −58° C. After 30 minutes, a solution ofα-bromo-m-tolunitrile (23, 12.5 g, 63.9 mmol) and THF (50 mL) was addedover 30 seconds (temperature rose from −78 to −60° C.). The cooling bathwas immediately replaced by a ice/H₂O bath and the reaction solution wasmaintained at ˜0° C. for 20 minutes. The reaction solution was quenchedwith 1 N HCl (100 mL). The layers were separated and the organic layerwas washed with brine (100 mL). The organic layer was dried (MgSO₄),filtered, and concentrated. The resulting residue was purified by flashcolumn chromatography (10:1 hexanes:EtOAc) to yield 11.6 g (60%) of 24.Inspection of the ¹H NMR revealed that 24 was only ˜80% pure, but nofurther purification was performed. LRMS (MH-tBuOCO) m/z 231.1.

Lithium bis(trimethylsilyl)amide (LHMDS, 1.0 M in THF, 28.4 mL, 3.3equiv) was added slowly via syringe to a −78° C. solution of ketone 24(2.84 g, 8.6 mmol) and THF (40 mL). The reaction solution temperaturewas monitored by an internal thermometer, and addition of the base wasdone at a rate sufficient to prevent the temperature from exceeding −48°C. After the addition was complete the resulting solution was maintainedat −78° C. for 30 mins. Neat 4-chloro-2-fluorobenzoyl chloride (18, 1.38mL, 8.6 mmol) (assumed density to be 1.20 g/mL) was added dropwise viasyringe. The reaction solution turned to an orange color and wasmaintained for 30 mins. The reaction solution was quenched with 1 N HCl(20 mL). The layers were separated and the organic layer was washed withbrine (20 mL). The organic layer was dried (MgSO₄), filtered, andconcentrated. The resulting residue was used without furtherpurification.

A mixture of the above crude product, K₂CO₃ (1.43 g, 10.34 mmol) and DMF(43 mL) was maintained at 23° C. for 1 hour. The reaction mixture wasquenched by addition of Et₂O (100 mL) and brine (200 mL). The layerswere separated and the organic layer was washed with brine (2×200 mL).The organic layer was dried (MgSO₄), filtered, and concentrated. Theresulting residue absorbed onto silica gel (CH₂Cl₂) and was purified byflash column chromatography (5:1 hexanes:EtOAc) to yield 2.7 g (67% for2 steps) of 25. Inspection of the ¹H NMR revealed that 25 was only ˜80%pure, but no further purification was performed. LRMS (MH) m/z 467.1.

Chromenone 25 (2.71 g, 5.80 mmol) and TFA:H₂O (97.5:2.5, 25 mL) wasmaintained at 23° C. for 1 h. The reaction mixture was concentrated. Theresidue was dissolved in EtOAc (100 mL) and washed with 1 N NaOH (25 mL)and brine (25 mL). The organic layer was dried (MgSO₄), filtered, andconcentrated. The resulting residue was purified by flash columnchromatography (1:1 hexanes:EtOAc; 1:2 hexanes:EtOAc; 1:4 hexanes:EtOAc)to yield 1.20 g (56%) of 26. LRMS (MH) m/z 367.1.

Example 6

Lithium bis(trimethylsilyl)amide (LHMDS, 1.0 M in THF, 23.5 mL, 3.3equiv) was added slowly via syringe to a −78° C. solution of ketone 17(2.18 g, 7.14 mmol) and THF (20 mL). The reaction solution temperaturewas monitored by an internal thermometer, and addition of the base wasdone at a rate sufficient to prevent the temperature from exceeding −50°C. After the addition was complete the resulting solution was maintainedat −78° C. for 30 mins. Neat 2,4-difluorobenzoyl chloride (27, 1.05 mL,8.57 mmol) was added dropwise via syringe. The reaction solution turnedto an orange color and was maintained for 30 mins. The reaction solutionwas quenched with 1 N HCl (20 mL). The layers were separated and theorganic layer was washed with brine (20 mL). The organic layer was dried(MgSO₄), filtered, and concentrated. The resulting residue was purifiedby flash column chromatography (10:1 hexanes:EtOAc) to yield 2.16 g(68%) of 28. Inspection of the ¹H NMR revealed that 28 was only ˜85%pure, but no further purification was performed.

A mixture of 28 (2.16 g, 4.9 mmol), K₂CO₃ (812 mg, 5.86 mmol), and DMF(24 mL) was maintained at 23° C. for 1 hour. The reaction mixture wasquenched with brine (50 mL), and diluted with Et₂O (50 mL). The layerswere separated and the organic layer was washed with brine (2×50 mL).The organic layer was dried (MgSO₄), filtered, and concentrated. Theresulting residue was purified by flash column chromatography (10:1hexanes:EtOAc) to yield 1.25 g (60%) of 29. LRMS (MH) m/z 426.2.

Example 7

Lithium bis(trimethylsilyl)amide (LHMDS, 1.0 M in THF, 47 mL, 3.3 equiv)was added slowly via syringe to a −78° C. solution of ketone 17 (4.37 g,14.31 mmol) and THF (55 mL). The reaction solution temperature wasmonitored by an internal thermometer, and addition of the base was doneat a rate sufficient to prevent the temperature from exceeding −50° C.After the addition was complete the resulting solution was maintained at−78° C. for 30 mins. A solution of 4-cyano-2-fluorobenzoyl chloride (30,1.38 mL, 8.6 mmol) and THF (5 mL) was added rapidly via syringe(temperature rose from −78 to −50° C.). The reaction solution turned toa dark red color and was maintained for 30 mins. The reaction solutionwas quenched with 1 N HCl (20 mL). The layers were separated and theorganic layer was washed with brine (20 mL). The organic layer was dried(MgSO₄), filtered, and concentrated. The resulting residue was usedwithout further purification.

A mixture of the above crude product, K₂CO₃ (2.40 g, 17.17 mmol) and DMF(70 mL) was maintained at 23° C. of 1 hour. The reaction mixture wasquenched by addition of Et₂O (200 mL) and brine (200 mL). The layerswere separated and the organic layer was washed with brine (2×200 mL).The organic layer was dried (MgSO₄), filtered, and concentrated. Theresulting residue was purified by flash column chromatography (10:1hexanes:EtOAc) to yield 4.95 g (80% for 2 steps) of 31. LRMS (MH) m/z433.2.

Example 8

7-Fluoro-Chromenone 32 (96 mg, 0.19 mmol) was dissolved in 0.5 M sodiummethoxide in methanol (10 mL) and heated to 70° C. The temperature wasmaintained at 70° C. for 12 hours and then cooled to room temperature.The solvent was removed under reduced pressure. To the remaining residuewas added EtOAc (20 mL) and water (20 mL). The layers were separated andthe aqueous phase was extracted with additional EtOAc (3×15 mL). Theorganic phases were combined, washed with brine (25 mL) and dried(Na₂SO₄). Concentration in vacuo gave an amorphous white solid which waspurified using flash column chromatography (20:1 DCM:MeOH) to provide 92mg (94%) of 33. LRMS (MH) m/z 509.3.

Example 9

Compound 29 (608 mg, 1.43 mmol) was dissolved into a solution of 0.5 Msodium methoxide in methanol (40 mL). The mixture was heated to refluxfor 3 hours (or until LC-MS indicated no starting material left). Thenthe solvent was evaporated. The residue was dissolved in dichloromethane(200 mL) and water (200 mL) was added, in which pH of the mixed solutionwas adjusted to 9 by adding 2N HCl solution. The collected organiclayers were dried by sodium sulfate. After evaporation of solvents, thefinal compound 34 (580 mg, 92%) was dried under vacuum.

Example 10

To a solution of 32 (56 mg, 0.11 mmol) in DMF (5 mL) was added NaH (5 mgof 60% dispersion in mineral oil, 0.15 mmol). The resulting solution wasstirred at 45° C. for 5 mins., then allyl alcohol was added (10 μL, 0.15mmol) via pipette. The resulting solution was stirred at 45° C. for 12hours and then cooled to room temperature. EtOAc (50 mL) and water (15mL) were added and the layers were separated. The aqueous phase wasextracted with EtOAc (3×15 mL). The organic phases were combined, washedwith water (2×30 mL) and brine (2×30 mL), dried (Na₂SO₄) andconcentrated under reduced pressure to yield 35 as an amorphous solid,which was used without further purification (54 mg, 91%). LRMS (MH) m/z535.0.

To a room temperature solution of 35 (54 mg, 0.10 mmol) and MeCN (5 mL)was added morpholine (44 μL, 0.50 mmol), followed by Pd(PPh₃)₄ (5 mg,10%). The resulting solution was stirred for 5 minutes and thenconcentrated under reduced pressure. The remaining residue was purifiedby flash column chromatography using stepwise elution (20:1 DCM:MeOH,10:1 DCM:MeOH) to provide 41 mg of 36 as an off-white solid (81%). LRMS(MH) m/z 495.2.

Example 11 Preparation of3-Benzyl-7-chloro-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-chromen-4-onea){2-[1-(3-Benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propylamino]-ethyl}-carbamicacid tert-butyl ester

To a solution of2-(1-amino-2-methyl-propyl)-3-benzyl-7-chloro-chromen-4-one (6.0 g, 18mmol) and (3-oxoethyl)carbamic acid tert-butyl ester (3.6 g, 23 mmol) inmethylene chloride (150 mL) was added sodium triacetoxyborohydride (7.4g, 35 mmol). The reaction mixture was stirred at room temperature for 16hours, at which time it was diluted with 1 N sodium hydroxide (150 mL)and stirred vigorously for 2 hours. The organic layer was washed with 1N sodium hydroxide (100 mL) and brine (100 mL), dried over magnesiumsulfate, and concentrated. The residue was purified by flashchromatography (0→50% ethyl acetate/hexanes) to give 5.7 g (67%) of thetitle compound. MS (ES+) m/e 485 [M+H]⁺.

b)2-[1-(2-Amino-ethylamino)-2-methyl-propyl]-3-benzyl-7-chloro-chromen-4-one

A solution of{2-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propylamino]-ethyl}-carbamicacid tert-butyl ester (5.7 g, 12 mmol) in 4:1 methylenechloride/trifluoroacetic acid (250 mL) was maintained at roomtemperature for 1.5 hours, at which time it was concentrated. Theresidue was dissolved in methylene chloride (200 mL), washed with 10%sodium carbonate, saturated sodium bicarbonate, and brine, dried overmagnesium sulfate, and concentrated. The residue (4.2 g, 91% yield) wasused without further purification.

c)N-{2-[1-(3-Benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propylamino]-ethyl}-4-methyl-benzamide

To a cooled (0° C.) solution of2-[1-(2-amino-ethylamino)-2-methyl-propyl]-3-benzyl-7-chloro-chromen-4-one(4.1 g, 11 mmol) and triethylamine (2.2 mL, 17 mmol) in methylenechloride (100 mL) was added a solution of p-toluoyl chloride (1.7 g, 11mmol) in methylene chloride (20 mL). The reaction was maintained at 0°C. for 2 hours, at which time it was diluted with ether (250 mL). Theresultant solution was washed with 1 N hydrogen chloride (2×200 mL),saturated sodium bicarbonate (200 mL) and brine (150 mL), dried overmagnesium sulfate, and concentrated. The residue was purified by flashchromatography (0→60% ethyl acetate/hexanes) to give 2.8 g (50%) of thetitle compound. MS (ES+) m/e 503 [M+H]⁺.

d)3-Benzyl-7-chloro-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-chromen-4-one

A mixture ofN-{2-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propylamino]-ethyl}-4-methyl-benzamide(2.8 g, 5.5 mmol) and phosphorus oxychloride (9.4 mL, 100 mmol) intoluene (60 mL) was heated at 85° C. for 7 hours, then heated at refluxfor 1 hour. The reaction was concentrated and the residual phosphorusoxychloride removed by toluene azeotrope. The residue was diluted withethyl acetate (100 mL) and washed with saturated sodium bicarbonate (100mL) and brine (100 mL), dried over magnesium sulfate, and concentrated.The resultant residue was purified by flash chromatography (0→8%methanol/methylene chloride) to give 1.4 g (50%) of the title compound.MS (ES+) m/e 485 [M+H]⁺.

Following procedures analogous to those set forth herein in Examples 1,3, and/or 4, the following compounds were prepared:

R₇ R₁ R₉ [M + H]⁺ CN 3-MeO—Ph—CH₂— 3,4-piperonyl 536 Cl 3-MeO—Ph—CH₂—4-Me—Ph 515 Cl 3-MeO—Ph—CH₂— 3-F-4-Me—Ph 533 Cl 3-MeO—Ph—CH₂—3,4-piperonyl 545 F 3-MeO—Ph—CH₂— 4-Me—Ph 499 F 3-MeO—Ph—CH₂—3-F-4-Me—Ph 517

Example 12 Preparation of2-[(R)-1-(4-Aminomethyl-2-p-tolyl-imidazol-1-yl)-2-methyl-propyl]-3-benzyl-7-chloro-chromen-4-onea)2-[(R)-1-(3-Phthalimido-2-oxo-propylamino)-2-methyl-propyl]-3-benzyl-7-chloro-chromen-4-one

To 2-((R)-1-amino-2-methyl-propyl)-3-benzyl-7-chloro-chromen-4-one (0.5g, 1.5 mMol) and K₂CO₃ (0.21 g, 1.5 mMol) in DMF (10 mL) was addedN-(3-bromo-2-oxopropyl)-phthalimide (0.45 g, 1.5 mMol) (Nair et al.; J.Org. Chem.; 40; 1975; 1745). The reaction was stirred at RT for 3 h,concentrated under vacuum, taken up in EtOAc, washed with water, brine,dried (Na₂SO₄) and evaporated to give the title compound (0.88 g, 100%)as a yellow solid: MS (ES) m/e 543.2 (M+H)⁺.

b)2-{(R)-1-[N-Toluoyl-(3-phthalimido-2-oxo-propyl)amino]-2-methyl-propyl}-3-benzyl-7-chloro-chromen-4-one

To2-[(R)-1-(3-phthalimido-2-oxo-propylamino)-2-methyl-propyl]-3-benzyl-7-chloro-chromen-4-one(0.88 g, 1.5 mMol) in CH₂Cl₂ (10 mL) was added Et₃N (0.23 mL, 1.6 mMol)and toluoyl chloride (0.21 mL, 1.6 mMol). The reaction was stirred at RTfor 18 h, concentrated under vacuum, taken up in EtOAc, washed with 1 NHCl, brine, dried (MgSO₄) and evaporated to dryness. Purification byflash chromatography on silica gel (5-15% EtOAc/hexane) followed bytrituration with pet. ether, filtration and drying under vacuum gave thetitle compound (0.89 g, 90%) as a white solid: MS (ES) m/e 661.2 (M+H)⁺.

c)N-{1-[(R)-1-(3-Benzyl-7-chloro-4-oxo-chromen-2-yl)-2-methyl-propyl]-2-p-tolyl-1H-imidazol-4-ylmethyl}-phthalimide

To2-{(R)-1-[N-toluoyl-(3-phthalimido-2-oxo-propyl)amino]-2-methyl-propyl}-3-benzyl-7-chloro-chromen-4-one(0.88 g, 1.3 mMol) and NH₄OAc (5.13 g, 66.8 mMol) was added HOAc (15mL). The reaction was stirred and heated to reflux (155° C. oil bath)for 2.5 h, cooled to RT and concentrated under vacuum. The remainingresidue was taken up in EtOAc, washed with water, brine, dried (MgSO₄)and evaporated to dryness. Purification by flash chromatography onsilica gel (50% EtOAc/hexane) followed by trituration with (1:2) Et₂O/pet. ether, filtration and drying under vacuum gave the title compound(0.51 g, 61%) as a tan solid: ¹H NMR (400 MHz, CDCl₃) δ 8.11 (d, J=8.6Hz, 1H), 7.89 (2d, 2H), 7.73 (2d, 2H), 7.53 (d, J=1.8 Hz, 1H), 7.52 (s,1H), 7.39 (d, J=8.0 Hz, 2H), 7.36 (dd, 1H), 7.32 (d, J=8.0 Hz, 2H), 7.14(m, 3H), 6.74 (d, J=1.5 Hz, 1H), 6.72 (s, 1H), 5.04 (d, J=8.8 Hz, 1H),4.94 (s, 2H), 4.01 (d, J=15.5 Hz, 1H), 2.63 (m, 1H), 2.46 (s, 3H), 2.44(d, J=15.5 Hz, 1H), 0.95 (d, J=6.6 Hz, 3H), 0.29 (d, J=6.7 Hz, 3H); MS(ES) m/e 642.0 (M+H)⁺.

d)2-[(R)-1-(4-Aminomethyl-2-p-tolyl-imidazol-1-yl)-2-methyl-propyl]-3-benzyl-7-chloro-chromen-4-one

ToN-{1-[(R)-1-(3-benzyl-7-chloro-4-oxo-3,4-dihydro-chromen-2-yl)-2-methyl-propyl]-2-p-tolyl-1H-imidazol-4-ylmethyl}-phthalimide(0.50 g, 0.78 mMol) in EtOH (15 mL) was added hydrazine monohydrate(0.12 mL, 2.5 mMol). The reaction was stirred at RT for 72 h, filteredthrough a pad of Celite® to remove the insoluble precipitate, rinsedwith EtOH and evaporated to dryness. Purification by flashchromatography on silica gel [5-10% (5% NH₄OH in MeOH)/CH₂Cl₂] gave thetitle compound (364 mg, 91%) as a white solid foam: ¹H NMR (400 MHz,CDCl₃) δ 8.14 (d, J=8.6 Hz, 1H), 7.63 (d, J=1.8 Hz, 1H), 7.37 (m, 6H),7.15 (m, 3H), 6.71 (d, J=1.7 Hz, 1H), 6.69 (s, 1H), 5.00 (d, J=10.9 Hz,1H), 4.07 (d, J=15.5 Hz, 1H), 3.92 (d, 1H), 3.88 (d, 1H), 2.72 (br s,2H), 2.65 (m, 1H), 2.49 (s, 3H), 2.48 (d, J=15.5 Hz, 1H), 0.93 (d, J=6.6Hz, 3H), 0.30 (d, J=6.7 Hz, 3H); MS (ES) m/e 512.2 (M+H)⁺.

Following procedures analogous to those set forth above, the followingcompounds were prepared:

R₇ R₁ R₉ [M + H]⁺ F Ph—CH₂— 4-Me—Ph 510 F Ph—CH₂— 3-F-4-Me—Ph 528 CNPh—CH₂— 3-F-4-Me—Ph 535 Cl Ph—CH₂— 3-F-4-Me—Ph 544 Cl 3-MeO—Ph—CH₂—4-Me—Ph 556 Cl 3-MeO—Ph—CH₂— 3-F-4-Me—Ph 574 CN Ph—CH₂— 4-Me—Ph 517 Cl3-CN—Ph—CH₂— 4-Me—Ph 551 Cl 3-CN—Ph—CH₂— 3-F-4-Me—Ph 569 F 3-MeO—Ph—CH₂—4-Me—Ph 540 CN 3-MeO—Ph—CH₂— 4-Me—Ph 547 CN 3-CN—Ph—CH₂— 3-F-4-Me—Ph 560F 3-MeO—Ph—CH₂— 3-F-4-Me—Ph 588 F 3-CN—Ph—CH₂— 3-F-4-Me—Ph 553

Example 13 Preparation ofN-{1-[(R)-1-(3-Benzyl-7-chloro-4-oxo-3,4-dihydro-chromen-2-yl)-2-methyl-propyl]-2-p-tolyl-1H-imidazol-4-ylmethyl}-acetamide

To2-[(R)-1-(4-aminomethyl-2-p-tolyl-imidazol-1-yl)-2-methyl-propyl]-3-benzyl-7-chloro-chromen-4-one(0.17 g, 0.33 mMol) in CH₂Cl₂ (5 mL) was added with stirring, pyridine(27 uL, 0.33 mMol) and Ac₂O (63 uL, 0.67 mMol). After stirring at RT for4 h the reaction was concentrated under vacuum. The remaining residuewas triturated with (1:1) Et₂O/pet. ether, filtered and dried undervacuum to give the title compound (163 mg, 89%) as a white solid: ¹H NMR(400 MHz, CDCl₃) δ 8.14 (d, J=8.6 Hz, 1H), 7.59 (d, J=1.8 Hz, 1H), 7.43(m, 6H), 7.17 (m, 3H), 6.84 (br s, 1H), 6.71 (d, J=2.2 Hz, 1H), 6.69 (s,1H), 5.07 (d, J=10.9 Hz, 1H), 4.50 (2d, 1H), 4.43 (2d, 1H), 4.07 (d,J=15.5 Hz, 1H), 2.66 (m, 1H), 2.51 (s, 3H), 2.48 (d, J=15.5 Hz, 1H),2.00 (s, 3H), 0.95 (d, J=6.6 Hz, 3 H), 0.34 (d, J=6.7 Hz, 3H); MS (ES)m/e 554.4 (M+H)⁺.

Example 14

A mixture of 4-cyano-2-fluorobenzoic acid (1, 25 g), DMF (0.1 mL), andthionyl chloride (50 mL) in a 250 mL round-bottom flask equipped with areflux condenser under N₂ atmosphere was heated to reflux in a 90° C.oil bath for 30 mins. The reflux condenser was replaced with adistillation head and the excess thionyl chloride was distilled from thereaction pot. The oil bath temperature was increased to 120° C. tofacilitate distillation. After 2 hours, the reaction vessel was cooledto 23° C. and placed under reduced pressure (˜25 Torr). The reactionvessel was then heated to 90° C. for 20 mins, then to 130° C. for 90minutes to remove any excess remaining thionyl chloride. The reactionvessel was then cooled to 23° C. and placed under high vacuum (˜0.2Torr). The reaction vessel was equipped with a clean distillation headand collection flask. The vessel was placed into a 137° C. oil bath andthe product was distilled from the reaction pot (b.p.=107° C. @ ˜0.2Torr). Upon cooling the distillate solidified, providing 2 as a whitesolid in close to quantitative yield.

A mixture of 4-chloro-2-fluorobenzoic acid (3, 25 g), DMF (0.1 mL), andthionyl chloride (50 mL) in a 250 mL round-bottom flask equipped with areflux condenser under N₂ atmosphere was heated to reflux in a 90° C.oil bath for 30 mins. The reflux condenser was replaced with adistillation head and the excess thionyl chloride was distilled from thereaction pot. The oil bath temperature was increased to 120° C. tofacilitate distillation. After 2 hours, the reaction vessel was cooledto 23° C. and placed under reduced pressure (˜25 Torr). The reactionvessel was then heated to 90° C. for 20 mins, then to 120° C. for 90minutes to remove any excess remaining thionyl chloride. The reactionvessel was then cooled to 23° C. and then equipped with a cleandistillation head and collection flask. The vessel was placed into a160° C. oil bath and the product was distilled from the reaction pot(b.p.=124° C. @ ˜25 Torr), furnishing 4 was a colorless oil in nearquantitative yield.

Example 15

Piperonyloyl Chloride (1, 7.8 g, 42 mmol) was added to a solution of2-amino-2-methyl-1-propanol (4 ml, 42 mmol), CH₂Cl₂ (200 mL), andtriethylamine (11.7 ml, 84 mmol) at room temperature. The reactionsolution was concentrated after 45 minutes, and the resulting residuewas diluted with EtOAc (40 mL) and washed with brine (30 mL). Theorganic layer was dried (MgSO₄), filtered, and concentrated to yield alight brown oil. The crude material (2, 8.8 g) was used without furtherpurification.

Tetrapropylammonium perruthenate (TPAP, 638 mg, 1.8 mmol) was addedportion-wise to a solution of 2 (8.6 g, 36.3 mmol), CH₂Cl₂ (73 ml, 2mL/mmol), 4-methyl-morpholine N-oxide (6.4 g, 54.5 mmol), and molecularsieves, 4 Å activated powder (18 g, 500 mg/mmol) at 0° C. under N₂. Thereaction was allowed to warm to r.t. after 15 minutes. After 1 hour thereaction was complete (TLC) and was filtered through silica, eluted withEtOAc (100 mL), and the filtrate was concentrated. This yielded 7 g ofoff-white solid (3). The material was recrystallized: EtOAc (30 mL),MeOH (10 mL), and Hexanes (1 mL) were added portion wise with heatingand sonication. This was brought to a boil after which hexane (100 mL)was added while cooling. Crystals immediately began to precipitate asthe solution was cooled. The mixture was filtered and the crystals werewashed with hexane (10 mL) to provide 4.88 g (57%) of 3 as off-whitefluffy crystals.

Example 16

A mixture of 2-amino-2-methyl-1-propanol (4 ml, 42 mmol), CH₂Cl₂ (200mL), 4-acetyl benzoic acid (1, 6.9 g, 42 mmol), EDC(1-(3-dimethylaminopropyl)-3-eythylcarbodiimide HCl, 12.1 g, 63 mmol),HOBT (N-hydroxybenzotriazole H₂O, 6.4 g, 42 mmol), and Hunig's base(diisopropylethylamime, 22 mL, 126 mmol) was stirred at room temperaturefor 18 hours. Upon completion (TLC, LC/MS) the reaction mixture wasconcentrated, and the resulting residue was diluted with EtOAc (50 mL)and washed with NaHCO₃ (2×40 mL) and brine (40 mL). The organic layerwas dried (MgSO₄), filtered, and the filtrate was concentrated. Thecrude material was purified by flash column chromatography (4:1:1hexanes:EtOAc:CH₂Cl₂; 1:1:1 hexanes:EtOAc:CH₂Cl₂; 1:1 EtOAc:CH₂Cl₂) toremove bisacylated material. Alcohol 2 was obtained in 54% yield (5.36g).

Tetrapropylammonium perruthenate (TPAP, 387 mg, 1.1 mmol) was addedportion-wise to a solution of 2 (5.36 g, 22.8 mmol), CH₂Cl₂ (46 mL, 2mL/mmol), 4-methyl-morpholine N-oxide (4 g, 34.2 mmol), and molecularsieves, 4 Å activated powder (11.4 g, 500 mg/mmol) at 0° C. under N₂.The reaction was allowed to warm to r.t. after 15 minutes. After 1 hourthe reaction was complete (TLC) and was filtered through silica, whichwas eluted with EtOAc (100 mL), and the filtrate was concentrated. Thisyielded 4.4 g of light pink solid (3). The material was recrystallized:1:1 Hexanes:EtOAc (5 mL), CH₂Cl₂ (20 mL), and MeOH (10 mL) were addedportion-wise with heating and sonication. This was brought to a boilafter which hexane (100 mL) was added while cooling. Crystalsimmediately began to precipitate as the solution was cooled. The mixturewas filtered and the crystals were washed with hexane (10 mL), affording2.46 g (46%) of 3 as off-white fluffy crystals.

Example 17

A solution of 4-acetyl benzoic acid (500 mg) in oxalyl chloride (5 mL)was heated to reflux for 2 hours. Any remaining oxalyl chloride wasevaporated by rot-yap, and the residue was dried by vacuum. The yield ofproduct was quantitative.

Example 18

((R)-1-Isopropyl-2-oxo-but-3-enyl)-carbamic acid tert-butyl ester

Tetrahydrofuran (THF, 100 mL) and a 1.0M solution of vinyl magnesiumbromide in THF (360 mL, 360 mmol, 3.1 equiv) was cooled to −78° C. whilestirring under a nitrogen atmosphere. The mixture was treated dropwisewith a solution of[(R)-(methoxy-methyl-carbamoyl)-methyl-propyl]-carbamic acid tert-butylester (30.3 g, 116 mmol, 1 equiv) in THF (50 mL) over 30 min. After theresultant dark yellow mixture was stirred for 30 min at −78° C., thecooling bath was removed and the reaction mixture was warmed slowly toroom temperature overnight (15 h). The reaction mixture was pouredslowly into an ice-chilled solution of 1N aqueous hydrochloric acid (700mL) and then warmed to room temperature. The organics were extractedwith (3×600 mL) ethyl acetate, dried over sodium sulfate, filtered, andconcentrated in vacuo. Purification by flash column chromatography(5-10% ethyl acetate/hexanes) provided the product as a white solid(16.8 g, 64%). ESMS [M+H]⁺: 228.4.

[(R)-(E)-1-Isopropyl-4-(3-methoxy-phenyl)-2-oxo-but-3-enyl]-carbamicacid tert-butyl ester

To a solution of ((R)-1-Isopropyl-2-oxo-but-3-enyl)-carbamic acidtert-butyl ester (13.54 g, 59.6 mmol) in dry acetonitrile (150 mL) underargon, was added 3-iodoanisole (13.96 g, 59.6 mmol), triethylamine (9.1mL, 65.6 mmol) followed by palladium (II) acetate (335 mg, 1.49 mmol).The resulting clear yellow solution was heated to 80° C. Upon heating,the reaction darkened and the precipitation of palladium black occurred.After 15 h, the reaction mixture was allowed to cool to roomtemperature, quenched with water (150 mL) and diluted with ether (150mL). The ether layer was washed with brine (100 mL) and the combinedaqueous layers were extracted with ether (two 50 mL portions). Theextracts were dried over magnesium sulfate, filtered and concentratedunder reduced pressure. The residue was immediately purified by silicagel chromatography (9:1 hexanes/EtOAc) to provide 17.6 g (88%) of[(R)-(E)-1-Isopropyl-4-(3-methoxy-phenyl)-2-oxo-but-3-enyl]-carbamicacid tert-butyl ester as a yellow oil. MS (ES+) m/e 334.0 [M+H]⁺.

[(R)—(Z)-4-(3-Cyano-phenyl)-1-isopropyl-2-oxo-but-3-enyl]-carbamic acidtert-butyl ester

Following the procedure described for[(R)-(E)-1-isopropyl-4-(3-methoxy-phenyl)-2-oxo-but-3-enyl]-carbamicacid tert-butyl ester with 3-iodobenzonitrile (5.50 g, 24.0 mmol, 1equiv) afforded the title compound as a yellow solid (7.4 g of ˜90%purity material). ESMS [M+H]⁺: 329.2.

[(R)-(E)-1-Isopropyl-4-(3-methoxy-phenyl)-2-oxo-butyl]-carbamic acidtert-butyl ester

To a solution of[(R)-(E)-1-Isopropyl-4-(3-methoxy-phenyl)-2-oxo-but-3-enyl]-carbamicacid tert-butyl ester (17.6 g, 52.9 mmol) in ethyl acetate (450 mL)under nitrogen was added 10 wt % palladium on carbon (300 mg). Thenitrogen was replaced with a balloon of hydrogen and the flask waspurged. After 3 h, the reaction flask was purged with nitrogen andfiltered through a pad of celite (rinsing with ethyl acetate). Thefiltrate was concentrated under reduced pressure and the residue waspurified by silica gel chromatography (9:1 hexanes/EtOAc) to provide16.2 g (91%) of[(R)-(E)-1-Isopropyl-4-(3-methoxy-phenyl)-2-oxo-butyl]-carbamic acidtert-butyl ester as a colorless oil. MS(ES+) m/e 336.4 [M+H]⁺. [α]_(D)²⁰=+19.1 (c=0.755, MeOH). ¹H NMR (400 MHz, CDCl₃) δ 7.21 (m, 1H),6.80-6.77 (m, 2H), 6.75 (s, 1H), 5.13 (d, J=8.4 Hz, 1H), 4.28 (dd,J=8.8, 4.4 Hz, 1H), 3.81 (s, 3H), 2.93-2.88 (m, 2H), 2.85-2.76 (m, 2H),2.14 (m, 1H), 1.46 (s, 9H), 1.00 (d, J=6.8 Hz, 3H), 0.75 (d, J=6.8 Hz,3H).

[(R)-4-(3-Cyano-phenyl)-1-isopropyl-2-oxo-butyl]-carbamic acidtert-butyl ester

Palladium on carbon (740 mg of 10% wt/wt Pd/C) was added to a degassedsolution of[(R)—(Z)-4-(3-cyano-phenyl)-1-isopropyl-2-oxo-but-3-enyl]-carbamic acidtert-butyl ester (7.4 g, 22.5 mmol, 1 equiv) in methanol (200 mL) atroom temperature. The reaction mixture was thoroughly degassed andbackfilled with hydrogen gas from a balloon. Hydrogenation proceeded atatmospheric pressure for 2.5 h. The (degassed) reaction mixture was thendiluted with diethyl ether (300 mL), filtered through Celite, and washedwith additional ether (2×100 mL). Upon concentration in vacuo, theresidue was purified by flash column chromatography (20% ethylacetate-hexanes) to provide the ketone product as a white solid (5.9 g,79%). ESMS [M+H]⁺: 331.2. ¹H NMR (400 MHz, CDCl₃) δ 7.51 (m, 2H), 7.46(m, 1H), 7.41 (m, 1H), 5.07 (m, 1H), 4.24 (m, 1H), 2.98 (m, 2H), 2.86(m, 2H), 2.11 (m, 1H), 1.45 (s, 9H), 0.98 (d, 3H, J=6.76 Hz), 0.74 (d,3H, J=6.78 Hz). [α]_(D)=+24.74 (c=0.95, CH₃OH).

Example 19

Following procedures analogous to those set forth above, the followingcompounds were prepared:

R₇ R₁ R₃ [M + H]⁺ F 3-MeO—Ph—CH₂— 3-F-4-Me—Ph— 549 F 3-MeO—Ph—CH₂—4-MeO—Ph— 547 F 3-MeO—Ph—CH₂— 4-Me—Ph— 531 F 3-MeO—Ph—CH₂—3,4-piperonyl- 561 F 3-MeO—Ph—CH₂— Methoxymethyl- 485 CN 3-MeO—Ph—CH₂—4-Ac—Ph— 566 CN 3-MeO—Ph—CH₂— 4-Me—Ph— 538 CN 3-MeO—Ph—CH₂—3,4-piperonyl- 568 CN 3-MeO—Ph—CH₂— 3-F-4-Me—Ph— 556 Cl 3-MeO—Ph—CH₂—3-F-4-Me—Ph— 565 Cl 3-MeO—Ph—CH₂— 4-Me—Ph— 547 Cl 3-MeO—Ph—CH₂—Methoxymethyl- 501 Cl 3-MeO—Ph—CH₂— 3,4-piperonyl- 577 MeO—3-MeO—Ph—CH₂— 3-F-4-Me—Ph— 561 MeO— 3-MeO—Ph—CH₂— 4-Me—Ph— 543 MeO—3-MeO—Ph—CH₂— 3,4-piperonyl 573 OH 3-MeO—Ph—CH₂— 3-F-4-Me—Ph— 547 CNPh—CH₂— 4-OEt—Ph— 538.2 CN Ph—CH₂— 6-trifluoromethyl-3-pyridyl 563.2 CNPh—CH₂— 2-benzo[b]thiophene 550.2 CN Ph—CH₂— 3-(5-t-butyl-2-methyl-2H-554.4 pyrazole) CN Ph—CH₂— 3-(2,5-dimethyl-2H-pyrazole) 512.4 F Ph—CH₂—6-trifluoromethyl-3-pyridyl 556.0 F Ph—CH₂— 2-furyl 477.0 F Ph—CH₂—3-(5-t-butyl-2-methyl-2H- 547.2 pyrazole) Cl Ph—CH₂— 4-CN—Ph— 529.2 ClPh—CH₂— 4-AcNH—Ph— 561.4 Cl Ph—CH₂— 6-trifluoromethyl-3-pyridyl- 573.0Cl Ph—CH₂— 5-benzo[1,2,3]thiadiazole- 562.2 Cl Ph—CH₂— 2-furyl- 494.4 ClPh—CH₂— 3-(2,5-dimethyl-2H-pyrazole)- 522.0 F Ph—CH₂— 4-pyridyl- 488.4 FPh—CH₂— 3-(2,5-dimethyl-2H-pyrazole)- 505.4 F Ph—CH₂— 4-AcNH—Ph— 544.4 FPh—CH₂— 5-benzo[1,2,3]thiadiazole- 545.2 CN Ph—CH₂—2-(1-methyl-1H-indole)- 547.4 Cl Ph—CH₂— 3-pyridyl- 505.2 F Ph—CH₂—4-OMe—Ph— 517.2 CN Ph—CH₂— 3-(2,5-dimethyl-furan)- 512.2 F Ph—CH₂—3-(2,5-dimethyl-furan)- 505.2 CN Ph—CH₂— 2-(5-methyl-thiophene)- 514.2 FPh—CH₂— 2-(5-methyl-thiophene)- 507.0 CN Ph—CH₂—2-(1-methyl-1H-pyrrole)- 497.4 CN Ph—CH₂— 5-benzo[1,2,3]thiadiazole-550.0 Cl Ph—CH₂— 6-Me-3-pyridyl- 518.2 Cl Ph—CH₂—2-(1-methyl-1H-pyrrole)- 506.0 Cl Ph—CH₂— 2-(5-methyl-pyrazine)- 520.2Cl Ph—CH₂— 3-(5-methyl-isoxazole)- 508.2 Cl Ph—CH₂— 3-benzo[c]isoxazole-544.2 CN Ph—CH₂— 4-(1-methyl-1H-imidazole)- 498.1 CN Ph—CH₂—3-N(CH₃)₂—Ph— 537.4 CN Ph—CH₂— 3-(5-methyl-2-trifluoromethyl- 566.4furan)- CN Ph—CH₂— 3-(5-methyl-isoxazole)- 499.6 F Ph—CH₂—4-(1-methyl-1H-imidazole)- 491.4 F Ph—CH₂— 2-(1-methyl-1H-pyrrole)-490.4 F Ph—CH₂— 3-benzo[c]isoxazole- 528.4 F Ph—CH₂—3-(5-methyl-isoxazole)- 492.4

Example 20

A pharmaceutical composition for intravenous administration is preparedin the following manner.

1 mg/mL (as free base) IV solution with the vehicle being pH 5.0, 50 mMsodium acetate buffer containing 3.5% (w/v) mannitol:

Composition* Unit Formula (mg/mL) Compound of Example 2 1.000 (freebase) Glacial Acetic Acid 1.081 Sodium Acetate Trihydrate 4.355Mannitol, pyrogen free 35.000 Water for Injection (WFI) q.s. to 1 mL*All components other than the active compound are USP or Ph. Eur.

A suitable compounding vessel is filled to approximately 75% of the bulksolution volume with WFI. The glacial acetic acid (1.081 g), sodiumacetate trihydrate (4.355 g), mannitol (35.000 g), and active (1.000 g)are weighed and individually added to the compounding vessel. After theadditions, the ingredients are dissolved in the mixture by stirring witha mixer. The pH of the bulk solution is measured and adjusted to 5.0with 5N NaOH or 5N glacial acetic acid. The solution is brought to itsfinal volume (1 liter) with WFI. Where the active compound is apharmaceutically acceptable salt, hydrate or salt of a hydrate, e.g., amonochloride salt or a hydrate of a monochloride salt, the amount of theactive equivalent to the free base is added.

Example 21 Preparation of a hydrochloride salt ofN-(3-Aminopropyl)-N-[(R)-1-3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamide

In one embodiment, a hydrochloric acid salt ofN-(3-Aminopropyl)-N-[(R)-1-3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamide,such as prepared in accordance with Example 2, is formed in thefollowing manner. An HCl salt is suitably prepared by reacting the freebase (e.g. dissolved in a suitable solvent, such as one or more of TBME,THF, and ethylacetate) with hydrochloric acid, suitably aqueoushydrochloric acid or otherwise in the presence of water. The mixture issuitably seeded with HCl salt to assist crystallization. The resultingsalt may be isolated using conventional techniques such as filtrationand drying (suitably washing the salt with a solvent, such as one ormore of TBME, THF, and ethylacetate).

In a particular embodiment, the salt is prepared as follows:

1. Dissolve the free base into 10 volumes of TBME and 5 volumes of THF,dissolution suitably conducted at room temperature;

2. Add aqueous HCl, e.g. 6.0 M or 12.0 M, suitably 1.1 eq, e.g.dropwise;

3. Suitably seed the solution with crystals of HCl salt, e.g. preparedaccording to preparation B below;

4. Suitably stir until crystals form;

5. Isolate the crystals, suitably by filtration, washing with a suitablesolvent such as TBME, and drying.

In more particular embodiments a salt is prepared as follows:

Preparation A

To a vial fitted with a stir bar add 500 mg ofN-(3-Aminopropyl)-N-[(R)-1-3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamide,and 3 ml TBME, and dissolve the free base in the TBME at roomtemperature. Bubble in HCl gas for about 30 seconds until a white solidforms. Filter and rinse the solids twice with 500 μl TBME. Transfer thesolids to a test tube, add 4 ml TBME and a stir bar, and stir at roomtemperature (the salt does not dissolve). Place the test tube containingthe mixture in an Argonaut RS10 heating block (Argonaut Technologies,Foster City, Calif.), or an equivalent, and temperature cycle asfollows:

1. 30 min at room temperature

2. ramp to 40 C over 30 min and hold at 40 C for 30 min

3. ramp to room temperature over 30 min and hold at room temperature for30 min

4. ramp to 10 C over 30 min and hold at 10 C for 30 min

5. ramp to room temperature over 30 min and hold at room temperature for30 min

6. repeat 2 to 5 as desired

By 20 hours cycling some solvent evaporates, a white powder forms on thesides and a melt-like solid forms in the bottom of the test tube. Add 2ml TBME. After 15 days, filter the light yellow solids and wash twicewith about 0.5 ml TBME then twice with 1 ml hexane. Dry in a vacuum ovenat room temperature for 12-24 hours to obtain about 440 mg HCl salt.

FIGS. 1 and 2 represent a XRPD and MDSC scan, respectively, of a saltprepared according to Preparation A.

Preparation B

Dissolve 4.23 g ofN-(3-Aminopropyl)-N-[(R)-1-3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamidein 5 volumes (21 ml) of THF. Add 10 volumes (42 ml) of TBME (tert-butylmethyl ether), then 1.1 eq of 12N HCl (750 μl) in one portion. Seed withHCl salt such as prepared in Preparation A (e.g. in 3 additions of about10 mg each staggered over about 10 minutes). Stir the mixture overnight,e.g. for about 12-24 hours. Filter the mixture, wash the solids withTBME, and dry the solids in a vacuum oven at room temperature overnight,e.g. for about 12-24 hours, to yield about 4.3 g of HCl salt.

FIG. 3 represents an XRPD scan of a salt prepared according toPreparation B.

The present invention includes crystallineN-(3-Aminopropyl)-N-[(R)-1-3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamidehydrochloride salt having characteristic X-ray diffraction peaks atabout 4.8, 9.7, 14.7, 17.9, 18.3, 20.1, 20.9, 22.5, 23.2, 23.8, 26.1 and26.9 degrees 2 theta.

Example 22

Inhibition of Cellular Viability in Tumor Cell Lines Treated with KSPInhibitors.r

Materials and Solutions:

-   -   Cells: SKOV3, Ovarian Cancer (human).    -   Media: Phenol Red Free RPMI+5% Fetal Bovine Serum+2 mM        L-glutamine.    -   Colorimetric Agent for Determining Cell Viability: Promega MTS        tetrazolium compound.    -   Control Compound for max cell kill: Topotecan, 1 μM.

Procedure: Day 1—Cell Plating:

Adherent SKOV3 cells are washed with 10 mLs of PBS followed by theaddition of 2 mLs of 0.25% trypsin and incubation for 5 minutes at 37°C. The cells are rinsed from the flask using 8 mL of media (phenolred-free RPMI+5% FBS) and transferred to fresh flask. Cell concentrationis determined using a Coulter counter and the appropriate volume ofcells to achieve 1000 cells/100 μL is calculated. 100 μL of media cellsuspension (adjusted to 1000 cells/100 μL) is added to all wells of96-well plates, followed by incubation for 18 to 24 hours at 37° C.,100% humidity, and 5% CO₂, allowing the cells to adhere to the plates.

Procedure: Day 2—Compound Addition:

To one column of the wells of an autoclaved assay block are added aninitial 2.5 μL of test compound(s) at 400× the highest desiredconcentration. 1.25 μL of 400× (400 μM) Topotecan is added to otherwells (optical density's from these wells are used to subtract out forbackground absorbance of dead cells and vehicle). 500 μL of mediawithout DMSO are added to the wells containing test compound, and 250 μLto the Topotecan wells. 250 μL of media+0.5% DMSO is added to allremaining wells, into which the test compound(s) are serially diluted.By row, compound-containing media is replica plated (in duplicate) fromthe assay block to the corresponding cell plates. The cell plates areincubated for 72 hours at 37° C., 100% humidity, and 5% CO₂.

Procedure: Day 4—MTS Addition and OD Reading:

The plates are removed from the incubator and 40 μl MTS/PMS is added toeach well. Plates are then incubated for 120 minutes at 37° C., 100%humidity, 5% CO₂, followed by reading the ODs at 490 nm after a 5 secondshaking cycle in a ninety-six well spectrophotometer.

Data Analysis

The normalized % of control (absorbance−background) is calculated and anXLfit is used to generate a dose-response curve from which theconcentration of compound required to inhibit viability by 50% isdetermined. The compounds of the present invention show activity whentested by this method as described above.

Example 23

Enantiomer Separation

An enriched 3:1 R:S mixture of chromenone enantiomers was separated intoits pure enantiomers by chiral chromatography with the followingconditions: Column—Chiralpak AD, 250×4.6 mm (Diacel Inc.). Sample—22.5mg/ml in 1:1 i-PrOH:hexanes. Conditions—40 min at isocratic 50% i-PrOHin Hexanes, (S)-enantiomer elutes at 18.35 min, (R)-enantiomer elutes at26.87 min. The (R)-enantiomer was significantly more potent than the(S)-enantiomer of the compound of Example 2.

Example 24

Monopolar Spindle Formation Following Application of a KSP Inhibitor

Human tumor cells Skov-3 (ovarian) were plated in 96-well plates atdensities of 4,000 cells per well, allowed to adhere for 24 hours, andtreated with various concentrations of the chromenone compounds for 24hours. Cells were fixed in 4% formaldehyde and stained with antitubulinantibodies (subsequently recognized using fluorescently-labeledsecondary antibody) and Hoechst dye (which stains DNA).

Visual inspection revealed that the compounds caused cell cycle arrestin the prometaphase stage of mitosis. DNA was condensed and spindleformation had initiated, but arrested cells uniformly displayedmonopolar spindles, indicating that there was an inhibition of spindlepole body separation. Microinjection of anti-KSP antibodies also causesmitotic arrest with arrested cells displaying monopolar spindles.

Example 25

Inhibition of Cellular Proliferation in Tumor Cell Lines Treated withKSP Inhibitors.

Cells were plated in 96-well plates at densities from 1000-2500cells/well of a 96-well plate and allowed to adhere/grow for 24 hours.They were then treated with various concentrations of drug for 48 hours.The time at which compounds are added is considered T₀. Atetrazolium-based assay using the reagent3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) (U.S. Pat. No. 5,185,450) (see Promega product catalog #G3580,CellTiter 96® AQ_(ueous) One Solution Cell Proliferation Assay) was usedto determine the number of viable cells at T₀ and the number of cellsremaining after 48 hours compound exposure. The number of cellsremaining after 48 hours was compared to the number of viable cells atthe time of drug addition, allowing for calculation of growthinhibition.

The growth over 48 hours of cells in control wells that had been treatedwith vehicle only (0.25% DMSO) is considered 100% growth and the growthof cells in wells with compounds is compared to this.

A Gi₅₀ was calculated by plotting the concentration of compound in μM vsthe percentage of cell growth in treated wells. The Gi₅₀ calculated forthe compounds is the estimated concentration at which growth isinhibited by 50% compared to control, i.e., the concentration at which:100×[(Treated₄₈ −T ₀)/(Control₄₈ −T ₀)]=50wherein Treated₄₈ is the value at 48 hours for the treated cells andControl₄₈ is the value at 48 hours for the control population.

All concentrations of compounds are tested in duplicate and controls areaveraged over 12 wells. A very similar 96-well plate layout and Gi₅₀calculation scheme is used by the National Cancer Institute (see Monks,et al., J. Natl. Cancer Inst. 83:757-766 (1991)). However, the method bywhich the National Cancer Institute quantitates cell number does not useMTS, but instead employs alternative methods.

Compounds of Examples 1-13 above inhibited cell proliferation in humanovarian tumor cell lines (SKOV-3).

Example 26

Calculation of IC₅₀:

Measurement of a compound's IC₅₀ for KSP activity uses an ATPase assay.The following solutions are used: Solution 1 consists of 3 mMphosphoenolpyruvate potassium salt (Sigma P-7127), 2 mM ATP (SigmaA-3377), 1 mM IDTT (Sigma D-9779), 5 μM paclitaxel (Sigma T-7402), 10ppm antifoam 289 (Sigma A-8436), 25 mM Pipes/KOH pH 6.8 (Sigma P6757), 2mM MgCl2 (VWR JT400301), and 1 mM EGTA (Sigma E3889). Solution 2consists of 1 mM NADH (Sigma N8129), 0.2 mg/ml BSA (Sigma A7906),pyruvate kinase 7 U/ml, L-lactate dehydrogenase 10 U/ml (Sigma P0294),100 nM KSP motor domain, 50 μg/ml microtubules, 1 mM DTT (Sigma D9779),5 μM paclitaxel (Sigma T-7402), 10 ppm antifoam 289 (Sigma A-8436), 25mM Pipes/KOH pH 6.8 (Sigma P6757), 2 mM MgCl2 (VWR JT4003-01), and 1 mMEGTA (Sigma E3889). Serial dilutions (8-12 two-fold dilutions) of thecompound are made in a 96-well microtiter plate (Corning Costar 3695)using Solution 1. Following serial dilution each well has 50 μl ofSolution 1. The reaction is started by adding 50 μl of solution 2 toeach well. This may be done with a multichannel pipettor either manuallyor with automated liquid handling devices. The microtiter plate is thentransferred to a microplate absorbance reader and multiple absorbancereadings at 340 nm are taken for each well in a kinetic mode. Theobserved rate of change, which is proportional to the ATPase rate, isthen plotted as a function of the compound concentration. For a standardIC₅₀ determination the data acquired is fit by the following fourparameter equation using a nonlinear fitting program (e.g., Grafit 4):

$y = {\frac{Range}{1 + \left( \frac{x}{{IC}_{50}} \right)^{s}} + {Background}}$where y is the observed rate and x is the compound concentration.

What is claimed is:
 1. A method of treating Hodgkin's disease,comprising administering to a patient in need of such treatment aneffective amount ofN-(3-aminopropyl)-N—[(R)-(1-3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamide,pharmaceutically acceptable salt thereof, a solvate thereof, or asolvate of a pharmaceutically acceptable salt thereof.
 2. A method oftreating Hodgkin's disease, comprising administering to a patient inneed of such treatment an effective amount ofN-(3-aminopropyl)-N—[(R)-(1-3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamidehydrochloride or a solvate thereof.
 3. A method of treating Hodgkin'sdisease, comprising administering to a patient in need of such treatmentan effective amount ofN-(3-aminopropyl)-N—[(R)-(1-3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamidehydrochloride hydrate.
 4. The method of claim 3, wherein theN-(3-aminopropyl)-N—[(R)-(1-3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamidehydrochloride hydrate has powder XRPD peaks (2θ) chosen from thosehaving approximately the following values: 4.8, 9.7, 20.0, and 23.7. 5.A method of treating non-Hodgkin's lymphoma, comprising administering toa patient in need of such treatment an effective amount ofN-(3-aminopropyl)-N—[(R)-(1-3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamide,a pharmaceutically acceptable salt thereof, a solvate thereof, or asolvate of a pharmaceutically acceptable salt thereof.
 6. A method oftreating non-Hodgkin's lymphoma, comprising administering to a patientin need of such treatment an effective amount ofN-(3-aminopropyl)-N—[(R)-(1-3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamidehydrochloride or a solvate thereof.
 7. A method of treatingnon-Hodgkin's lymphoma, comprising administering to a patient in need ofsuch treatment an effective amount ofN-(3-aminopropyl)-N—[(R)-(1-3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamidehydrochloride hydrate.
 8. The method of claim 7, wherein theN-(3-aminopropyl)-N—[(R)-(1-3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamidehydrochloride hydrate has powder XRPD peaks (2θ) chosen from thosehaving approximately the following values: 4.8, 9.7, 20.0, and 23.7.